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Critical to the success of human corneal transplants is prevention of corneal endothelial rejection, yet little is known about the endothelial infiltrate. To examine the endothelium, a method for removal and processing this layer as a flat sheet was used and the infiltrate was compared with stroma and epithelium. LEW or PVG strain rat corneas were transplanted to PVG strain recipients. Clinical changes after transplantation were monitored by slit lamp and animals sacrificed at a range of time points during rejection. Clinically defined rejection, accompanied by an epithelial rejection line and endothelial cell infiltration, occurred between days 10 and 15. There was some infiltration of leukocytes in the stroma of isografts at these time points, but significantly more in allografts (p

The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas.The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents.Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen.The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.

AIM—To examine the outcome of care for patients with glaucoma followed up by the hospital eye service compared with those followed up by community optometrists. METHODS—A randomised study with patients allocated to follow up by the hospital eye service or community optometrists was carried out in the former county of Avon in south west England. 403 patients with established or suspected primary open angle glaucoma attending Bristol Eye Hospital and meeting defined inclusion and exclusion criteria were studied. The mean number of missed points on visual field testing in the better eye (using a "better/worse" eye analysis) in each group were measured. The visual field was measured using the Henson semiautomated central field analyser (CFA 3000). Measurements were made by the research team on all patients at baseline before randomisation and again 2 years after randomisation. The mean number of missed points on visual field testing in the worse eye, mean intraocular pressure (mm Hg), and cup disc ratio using a "better/worse" eye analysis in each group at 2 years were also measured. Measurements were made by the research team on all patients at baseline before randomisation and again 2 years after randomisation. An analysis of covariance comparing method of follow up taking into account baseline measurements of outcome variables was carried out. Additional control was considered for age, sex, diagnostic group (glaucoma suspect/established primary open angle glaucoma), and treatment (any/none). RESULTS—From examination of patient notes, 2780 patients with established or suspected glaucoma were identified. Of these, 752 (27.1%) fulfilled the entry criteria. For hospital and community follow up group respectively, mean number of missed points on visual field testing at 2 year follow up for better eye was 7.9 points and 6.8 points; for the worse eye 20.2 points and 18.4 points. Similarly, intraocular pressure was 19.3 mm Hg and 19.3 mm Hg (better eye), and 19.1 mm Hg and 19.0 mm Hg (worse eye); cup disc ratio at 2 year follow up was 0.72 and 0.72 (better eye), and 0.74 and 0.74 for hospital and community follow up group respectively. No significant differences in any of the key visual variables were found between the two groups before or after adjusting for baseline values and age, sex, treatment, and type of glaucoma. CONCLUSIONS—It is feasible to set and run shared care schemes for a proportion of patients with suspected and established glaucoma using community optometrists. After 2 years (a relatively short time in the life of a patient with glaucoma), there were no marked or statistically significant differences in outcome between patients followed up in the hospital eye service or by community optometrists. Decisions to implement such schemes need to be based on careful consideration of the costs of such schemes and local circumstances, including geographical access and the current organisation of glaucoma care within the hospital eye service.

AIMS To investigate age related alterations in the non-collagenous components of the human lamina cribrosa. METHODS Fibronectin, elastin, and glial fibrillary acidic protein (GFAP) staining were assessed in young and old laminae cribrosae. An age range (7 days to 96 years) of human laminae cribrosae were analysed for lipid content (n=9), cellularity (n=28), total sulphated glycosaminoglycans (n=28), elastin content (n=9), and water content (n=56), using chloroform-methanol extraction, fluorimetry, the dimethylmethylene blue assay, and ion exchange chromatography, respectively. RESULTS Qualitatively, an increase in elastin and a decrease in fibronectin and GFAP were demonstrated when young tissue was compared with the elderly. Biochemical analysis of the ageing human lamina cribrosa demonstrated that elastin content increased from 8% to 28% dry tissue weight, total sulphated glycosaminoglycans decreased, and lipid content decreased from 45% to 25%. There were no significant changes in total cellularity or water content. CONCLUSION These alterations in composition may be indicative of the metabolic state of the lamina cribrosa as it ages, and may contribute to changes in mechanical integrity. Such changes may be implicated in the susceptibility of the elderly lamina cribrosa and also its response to glaucomatous optic neuropathy.

AIMS To investigate changes in the mechanical compliance of ex vivo human lamina cribrosa with age. METHODS A laser scanning confocal microscope was used to image the surface of the fluorescently labelled lamina cribrosa in cadaver eyes. A method was developed to determine changes in the volume and strain of the lamina cribrosa created by increases in pressure. The ability of the lamina cribrosa to reverse its deformation on removal of pressure was also measured. RESULTS Volume and strain measurements both demonstrated that the lamina cribrosa increased in stiffness with age and the level of pressure applied. The ability of the lamina cribrosa to regain its original shape and size on removal of pressure appeared to decrease with age, demonstrating an age related decrease in resilience of the lamina cribrosa. CONCLUSIONS The mechanical compliance of the human lamina cribrosa decreased with age. Misalignment of compliant cribriform plates in a young eye may exert a lesser stress on nerve axons, than that exerted by the rigid plates of an elderly lamina cribrosa. The resilience of the lamina cribrosa also decreased with age, suggesting an increased susceptibility to plastic flow and permanent deformation. Such changes may be of importance in the explanation of age related optic neuropathy in primary open angle glaucoma.

The effects of intravenous administration of nedocromil sodium were investigated in active and passive models of conjunctival immediate hypersensitivity in rats. In the active sensitization model, animals were immunized with ovalbumin 21 days prior to ocular instillation of a solution containing ovalbumin. Nedocromil sodium administered prior to antigen challenge significantly inhibited emergence of conjunctival edema and erythema (P < .05) and reduced mast cell degranulation (P < .02). In the passive-sensitization model, the conjunctiva in one eye was injected with ovalbumin antiserum 48 hours prior to intravenous administration of ovalbumin. Nedocromil sodium administered prior to antigen challenge significantly and dose-dependently reduced appearance of the signs of conjunctivitis (P < .01) as well as vascular leakage (P < .05). These data indicate that intravenous nedocromil sodium is effective in animal models of allergic conjunctivitis and may have potential for wider therapeutic application. These data are also consistent with results of clinical studies in which nedocromil sodium relieved symptoms of allergic conjunctivitis and further support a role for nedocromil sodium in the prevention of allergic conjunctivitis.

The ability of herpes simplex virus (HSV-1) to reactivate from latency in sensory ganglia is central to the pathogenesis of recurrent infection. Several studies have strongly suggested that the neuron is the site of reactivation (16, 21), although the fate of such neurons is still undetermined. After reactivation in vivo in the trigeminal ganglion (TG), only small numbers of virus antigen-positive neurons have been identified, and only small amounts of infectious virus were detected (16, 21). This highly restricted replication may explain the failure to detect virus DNA replication and the transience of expression of productive-cycle transcripts (1). Reactivation will, of course, occur in a host with an immunity already primed against the virus. It is therefore likely that the mounting of a virus-specific secondary immune response will play a major part in the rapid and effective control of infection. This supposition is supported by the observation of focal infiltrates of T cells, both CD4+ and CD8+, in close association with virus antigen-positive neurons as early as 1 day after stimulation to induce reactivation (21). Although these lymphocytes were the predominant infiltrating cell type when virus antigen was being cleared, by day 4 large numbers of B cells were also present, suggesting that local antibody production may also aid the control of reactivated infection. It appears that the efficiency of the immune system in controlling reactivated infection within the sensory ganglion, at least in the mouse, results in a significant proportion of reactivation events being aborted at an early stage, before they can lead to disease or viral shedding at the periphery (21). After reactivation, the initial presentation of antigen is likely to be mediated by resident major histocompatibility complex (MHC) class II+/F4/80+ immune cells (21). Their presence, together with the rapid appearance of T cells (probably virus-specific memory cells) provide the basis for the secondary immune response. However, a direct cytotoxic role for CD8+ T cells is problematic, since neurons do not normally express MHC class I and are well protected by ensheathing satellite cells. Nevertheless, these T cells may play a role in viral clearance via the production of antiviral cytokines. Evidence for such a function comes from studies on hepatitis B virus infection, where secretion of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), by CD8+ T cells can abolish viral gene expression and replication (7). The production of a range of cytokines in the TG following primary infection with HSV-1 has been investigated by a variety of methods, but there is no consensus on which cytokines are of primary importance during the clearance of virus. For example, using double staining we have demonstrated large numbers of TNF-α+ and/or IL-6+ cells, together with smaller numbers of IFN-γ+ cells, early in the course of infection (day 3), and these were seen in close association with virus antigen (22). mRNA for IFN-γ and TNF-α were detected by reverse-transcriptase (RT)-PCR at a similar time (3). In contrast, in the immunohistochemical study of Liu et al. (13) IFN-γ and IL-4 were the predominant cytokines present early in infection. The role of IL-10 during viral clearance also appears to be equivocal; in our studies no IL-10+ cells were detected, but others have identified small numbers of such cells (13) and Halford et al. (8) found mRNA for this cytokine in 100% of the TG samples taken 5 to 7 days after infection. In addition to infiltrating immune cells, the resident satellite and Schwann cells of the peripheral nervous system (PNS) and astrocytes in the central nervous system (CNS) at the dorsal root entry zone (DRE) can also be potent sources of TNF-α and IL-6. It has been suggested that, via these cytokines, glial cells may also be involved in viral clearance and in normal homeostatic mechanisms in the nervous system, such as repair and protection of neurons from damage (22). In contrast to the events in the TG during primary infection, the role of cytokines in reactivation has only recently started to be explored (4, 11, 14). We now report studies on cytokine production (IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α) in response to reactivated virus in the TG with a mouse model in which reactivation and recurrent disease are induced by UV irradiation of the cornea (18). The immunohistochemical method used allows simultaneous detection of virus antigen and cells producing the cytokines. During primary infection, virus can spread to the CNS via the DRE, resulting in immune cell infiltration and cytokine production at this site. To investigate whether, following reactivation of virus in the TG, similar events occur in the contiguous CNS, our tissue specimens included both the TG and its attached DRE (TG/DRE).

An improved method of removing rat corneal endothelial sheets for study of endothelial pathology is described. The method was validated by examining morphological changes and changes in expression of major histocompatibility complex (MHC) and intercellular adhesion molecule (ICAM)-1 on endothelium undergoing immunological rejection. PVG strain rats received LEW strain corneal transplants or corneal isografts. Just prior to and during graft rejection, animals were killed, together with a group of untreated animals. The corneal stroma was injected with dispase or PBS, the cornea was carefully removed, fixed in acetone and the endothelium was gently peeled off and flattened on to a glass slide. Morphological changes, together with MHC class I, class II and ICAM-1 expression were visualised by immuno-histochemical staining and quantified by image analysis. Near complete endothelial sheets were obtained by this method. Because of the thin cell layer, there was minimal background staining, permitting rejection-associated changes to be clearly seen. MHC class I expression on normal endothelium was low and not significantly increased on endothelial cells of allografts at the time of rejection compared with controls (P = 0.1). MHC class II and ICAM-1 were induced de novo, expression being significantly higher on allografts than on isografts (P = 0.004 for MHC class II and P = 0.01 for ICAM-1). MHC class I and II and ICAM-1 were expressed on many infiltrating cells. Thus, this preparation method permits clear identification of the distribution and morphology of infiltrating cells and other mediators of the immune response in the entire donor endothelium. It confirms that MHC class I expression is low during rejection, while MHC class II and ICAM-I are induced de novo and strongly expressed.

Objective To compare postoperative astigmatism induced by two different suturing techniques in penetrating keratoplasty (PKP). Design A monocenter, prospective, randomized clinical trial with a longitudinal 1-year follow-up. Participants A total of 95 eyes undergoing PKP were randomized into 2 groups. Of these, 51 eyes were allocated to the combined interrupted and continuous suturing group (ICS) and 44 eyes to the single continuous adjustable suturing (SCAS) group. Intervention In the ICS group, suturing was with a combination of 12 interrupted 10-0 nylon and 1 continuous 11-0 nylon sutures. Eyes in the SCAS group had been sutured with a single running 24-bite 10-0 nylon. Selective suture removal started no earlier than 10 weeks after surgery; suture adjustment could start as soon as possible after surgery. Main outcome measures Astigmatism was measured by topography, keratometry, and refraction at 3-, 6-, 9-, and 12-month postoperative intervals. Results The difference in mean time of suture manipulation between groups was significant ( P = 0.0001), with the SCAS starting earlier. A significant decrease in astigmatism occurred by either interrupted suture removal (6.69 ± 3.11 diopter [D] before to 4.76 ± 2.99 D after, P = 0.0002) or suture adjustment (7.18 ± 3.12 D before to 4.46 ± 3.24 D after, P = 0.0001). However, the net astigmatic reduction in the SCAS group was not significantly greater ( P = 0.250) than in the ICS group. Vector change was 7.40 ± 4.17 D and 6.28 ± 4.14 D for SCAS and ICS, respectively ( P = 0.13). At no interval (3, 6, 9, or 12 months) was there significant difference in astigmatism between the two groups. Refractive astigmatism (cyl, D) at 1 year was 2.66 ± 1.70 for the ICS and 3.12 ± 2.62 for the SCAS, but there was no significant treatment effect ( P = 0.945). Furthermore, 66% of the ICS eyes and 58% of the SCAS eyes ( P = 0.295) were within the astigmatic target of the study ( Conclusions Postkeratoplasty astigmatism can be decreased similarly with either adjustment of a single running suture or selective removal of interrupted sutures. No advantage of the SCAS over ICS in terms of fewer manipulations or less astigmatism was seen as suggested previously.

Objective To assess the effectiveness of computerized videokeratography (CVK) in refining the surgical design and in improving predictability of surgical correction of postkeratoplasty astigmatism. Design A prospective, controlled, randomized, clinical trial. Participants A total of 31 postkeratoplasty eyes, divided into 2 groups (group A, 16 eyes; group B, 15 eyes), with more than 4 diopters (D) of disabling astigmatism were studied. Intervention All eyes were treated with combination of arcuate relaxing incisions and compression sutures. The surgical plan in group A was based on topographic information, whereas in the control group B, the surgical plan was based on information obtained by refraction and keratometry alone. Main outcome measures Change in the surgical plan induced by the CVK information, astigmatism, topographic patterns, and factors associated with outcome were measured. Results In all 16 cases of group A, the use of CVK changed some aspect of the surgical plan. At 12 months after surgery, both groups showed a significant net reduction ( P = 0.001) of baseline astigmatism. However, the reduction (47% and 41% for groups A and B, respectively) did not differ significantly between the two groups. The topographic astigmatism at 12 months measured 4.24 ± 0.71 D in group A and 5.60 ± 0.51 D in group B ( P = 0.139). Significant differences between the two groups at 12 months were seen only for keratometric astigmatism (3.60 ± 0.81 D in group A vs. 5.77 ± 0.52 D in group B, P = 0.035) and refractive astigmatism (2.34 ± 0.37 D in group A vs. 4.88 ± 0.52 D in group B, P = 0.000). The mean vector surgical effect was 91% for group A and 70% for group B. Regular astigmatism patterns had a greater benefit from surgery than irregular patterns ( P = 0.008). Previous refractive surgery was associated with less-favorable outcome ( P = 0.045). Conclusions The current study indicates that the use of CVK provides a benefit compared to keratometry and refraction alone in the planning and outcome of surgical treatment for high postgraft astigmatism.

BACKGROUND The aims of the study were to determine (1) whether community optometrists are able to make valid measurements of visual parameters in patients with established or suspect primary open angle glaucoma and (2) patient satisfaction with follow-up by community optometrists. METHODS A randomized study was carried out in the former county of Avon in South West England with patients allocated to follow-up by the hospital eye service or by community optometrists. The subjects were 403 patients with established or suspected primary open angle glaucoma attending Bristol Eye Hospital and meeting defined inclusion and exclusion criteria. The main outcome measures were 91) measurements of key visual parameters (intraocular pressure, visual fields and cup/disc ratio) made by hospital eye service and community optometrists, compared with a research clinic reference standard at baseline, and (2) patient satisfaction at baseline and at six months. RESULTS Community optometrists were able to make measurements of comparable accuracy to those made in the hospital eye service. Patients were significantly more satisfied with a number of aspects of care provided by community optometrists, particularly those relating to waiting times, compared with those from the hospital eye service. CONCLUSIONS Community optometrists are able to make measurements of key visual parameters in patients with established or suspect primary open angle glaucoma which are of comparable quality to the hospital eye service. Follow-up by community optometrists is acceptable to patients. The costs of each option are reported elsewhere.

Purpose: Carbomer gel is a water-soluble polymeric resin that has been reported to maintain the tear film in contact with the eye for an extended period. The efficacy and safety of this new artificial tear were assessed. Methods: A multicenter, single-masked, randomized, placebo-controlled study was carried out on 123 patients with moderate-to-severe dry eyes. The placebo was a mannitol solution with benzalkonium chloride 0.008% as preservative. Patients were observed over an 8-week period, and subjective and objective changes analyzed, compared to a baseline of no therapy, after 1 to 7 days washout period from previous medication. Results: All primary subjective symptoms decreased significantly in the carbomer gel-treated group compared to the placebo group (i.e., dryness, discomfort, and foreign body sensation). The carbomer gel also significantly improved the rose bengal staining score relative to placebo. When data for the primary subjective efficacy variables were stratified for disease severity, there was a statistically significant improvement from baseline by day 10 for severely affected patients and from day 42 for patients with moderate disease. Secondary subjective symptoms that improved significantly in the tear gel group compared to placebo were photophobia, erythema, tear breakup time, blurry-filmy, drysandy sensation, and physician impression. However, no significant improvements in the secondary subjective symptoms of tearing, itching, scaling, conjunctival discharge, palpebral conjunctival redness, bulbar conjunctival redness, conjunctival luster, relief of discomfort, ease of use, and overall acceptability were found in either group over the baseline score. In addition, neither carbomer gel nor placebo improved the baseline fluorescein staining score or the Schirmer test score. Two patients suffered local allergic reactions to the carbomer gel or its preservative, which settled on withdrawal of the medication. Conclusions: Carbomer gel was more efficacious than was placebo in improving a number of subjective and objective symptoms of moderate-to-severe dry eye syndrome. The results of this study indicate that carbomer gel was as safe as was the placebo.

AIM—On the basis of finalised data from the Corneal Transplant Follow up Study to identify and quantify factors influencing corneal graft outcome in terms of graft survival, rejection, visual acuity, and astigmatism. METHODS—Multifactorial analysis of 2777 grafts registered by the UK Transplant Support Service from July 1987 to June 1991. RESULTS—Several recipient factors influencing graft survival, rejection, and visual acuity were identified, but no donor factors. Of the operative factors amenable to change, mixed suturing was associated with reduced graft survival, and larger grafts with increased risk of rejection but better visual acuity when surviving. There was increased risk of rejection with poor matching at HLA class I antigens, but mismatched HLA-DR grafts suffered less rejection than those with zero HLA-DR mismatches. Recipient age below 10 years was associated with increased risk of both rejection and graft failure. However, whereas increasing age above 10 years was not associated with differential graft survival, it was significantly associated with decreasing risk of rejection. CONCLUSIONS—While confirming possible benefits of HLA-A and B matching, the expense and delay involved in awaiting matched HLA-DR tissue is unlikely to be justified. Other donor factors are unrelated to graft outcome following screening of tissue by eye banks. The highest rates of graft failure and rejection happen in the early postoperative period, and factors influencing visual outcome are also apparent at this stage.

The corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4+ and CD8+. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.

In spite of improvements in surgical techniques, donor materials and postoperative care, high astigmatism remains a quite common problem following penetrating keratoplasty [1]. Whenever the residual astigmatism cannot be corrected with spectacles or contact lenses, surgical treatment is required. Relaxing incisions combined with compression sutures is one of the most common methods used for this purpose [2, 3]. We report herein a case of persistent aqueous leak following relaxing incisions for the correction of postkeratoplasty astigmatism. The leak failed to respond to a bandage contact lens and resuturing and was eventually successfully treated with the use of cyanoacrylate glue. A marked regression of the surgical effect was observed in this case.

Reactivation of latent herpes simplex virus type 1 (HSV-1) infection was induced by UV irradiation of the corneas of latently infected mice. On days 1-4 after stimulation, infectious virus was sought in nervous and ocular tissue. On days 4, 7 and 10, eyes with either recurrent epithelial or stromal disease and appropriate controls were stained to identify immune cells and HSV-1 antigens. The maximum incidence of infectious virus was on day 2 when 5/10 ophthalmic parts of the trigeminal ganglion yielded HSV. Thus in this mouse model, as in humans, reactivation of virus in the trigeminal ganglion is the likely source of virus producing recurrent disease and shedding in the tear film. On day 4, when virus antigens were still present, granulocytes were the predominant infiltrating cell in corneas with either type of disease. Small numbers of T cells, dendritic cells and cells expressing MHC class II were also present. In stromal disease, the granulocyte infiltrate persisted and T cells remained sparse. In contrast, in epithelial disease, granulocyte numbers rapidly declined and both CD4+ and CD8+ T cells (present at a ratio of 1:1) increased significantly. The secondary immune response to virus antigen is more rapid and vigorous than that during primary corneal infection. Granulocytes may play a role in the initial clearance of virus, however, the other types of cells present early on provide the potential for a local secondary immune response. The high proportion of CD8+ cells in epithelial disease compared with stromal disease suggests that they may be acting as suppressors.

AIM: To study the influence of cryoprotectant, cooling rate, and warming rate on recovery and viability of keratocytes from corneas for cryolathing. METHODS: Corneas were frozen at -50 degrees C for 2 minutes either after exposure to 10% dimethyl sulphoxide in Eagle's MEM for 15 minutes at room temperature (about 22 degrees C), or without earlier exposure to the cryoprotectant. Corneas were cooled either rapidly (20 degrees C/min) or slowly (1 degree C/min), and they were warmed either rapidly (> 50 degrees C/min) by direct transfer into medium at 22 degrees C or slowly (< 20 degrees C/min) in air at 22 degrees C. The cryoprotectant was removed by dilution in medium containing 0.5 mol/l sucrose. Recovery of keratocytes was determined by using collagenase digestion to release the cells from the stroma and trypan blue staining. Viability was assessed by the outgrowth of cells from stromal explants in primary tissue culture. RESULTS: The use of a cryoprotectant before freezing was beneficial, irrespective of the different cooling and warming regimens. Both collagenase digestion and tissue culture revealed that keratocyte survival was improved when corneas were warmed rapidly rather than slowly. The collagenase digestion assay showed an apparently higher recovery of keratocytes after slow cooling (54.3%) than after rapid cooling (34.1%), but no differences in cell viability could be demonstrated by primary tissue culture. CONCLUSION: Although in these experiments slow cooling apparently provided the best recovery of keratocyte numbers (though not viability), previous work had revealed some disruption of the epithelial basement membrane after slow cooling. If viable keratocytes and good preservation of epithelial basement membrane are considered to be prerequisites for epikeratophakia lenticules then it is suggested that corneas should be prepared for cryolathing by freezing rapidly after exposure to 10% dimethyl sulphoxide and, following cryolathing, they should be warmed rapidly.

Purpose: To quantify clinical and operative factors that influence corneal graft outcome. Methods: A multifactorial analysis was done on 2242 corneal grafts registered by the United Kingdom Transplant Service from July 1987, to June 1991. Results: There was an increased risk of graft failure in patients with preoperative diffuse and other noncentral stromal edema, less-common eye diseases, small trephine size, difference in donor and recipient sizes greater than 0.25 mm, and use of mixed continuous and interrupted sutures. Visual acuity 3 months after surgery was poorer in patients who had glaucoma and low visual acuity preoperatively, small trephine size, and combined vitreous surgery. Use of interrupted sutures resulted in higher astigmatism at 3 months. Conclusions: After allowing for the effects of recipient factors, surgical factors significantly affected corneal graft outcome. No factors that showed significant benefits for graft survival also adversely affected visual performance. Details of medical history, clinical condition, and surgical method failed to predict more than a small proportion of observed variability in visual performance of functioning grafts.

AIMS/BACKGROUND--This study was initiated to investigate risk factors for and outcome of Acanthamoeba keratitis. METHODS--Results of treatment were studied in 22 patients (23 eyes) presenting to Bristol Eye Hospital between 1985 and February 1995. Details related to the use and disinfection of contact lenses were also obtained. An additional two patients who were seen at Bristol but mainly treated elsewhere were surveyed for contact lens related information only. RESULTS--The incidence of Acanthamoeba keratitis rose substantially in the 1990s: three patients presented before 1990, while the remaining 21 presented between January 1990 and February 1995. Eleven patients have presented since january 1994. All of the patients in this series were contact lens wearers, 16 (67%) using daily wear disposable contact lenses. Contact lens disinfection data were available in 22 patients of whom 11 (50%) were using chlorine disinfectant. Other types of disinfection were much less common. Four patients (18%) had not used any disinfectant. During the course of the series the average diagnostic delay has fallen markedly, although in 77% of patients a diagnosis of a viral keratitis, most commonly herpes simplex, was made on first presentation. All but three of the series were treated with a combination of polyhexamethylene biguanide and propamidine isethionate. Penetrating keratoplasty was performed in 9/23 eyes (39%); in all of these eyes diagnosis was delayed for at least 6 weeks. All but one of the eyes in the series achieved a visual acuity of 6/9 or better after treatment, and 18 eyes (78%) saw 6/6 or better. CONCLUSIONS--Most patients with Acanthamoeba keratitis can now expect a good visual result and cure by medical therapy alone is favoured by early diagnosis.

The purpose of this study was the evaluation of community based optometric monitoring of stable glaucoma patients and glaucoma suspects compared to the routine Hospital Eye Services (HES) monitoring. Four hundred and five subjects were recruited from routine outpatient clinics at the Bristol Eye Hospital. All eligible participants were seen in the routine hospital clinic and then within two months were given a 'Gold Standard Assessment' (GSA) by an independent research team. Participants then visited one of 12 glaucoma-trained optometrists, for a standard battery of tests. Randomisation resulted in 204 subjects being allocated to community care, with reviews on a six monthly basis, with 201 who remained a control group within the hospital. Referral criteria were established to enable optometric detection of apparent glaucomatous progression. A questionnaire was used to assess patient satisfaction with both care types. Additionally a cost analysis exercise was performed.

Following inoculation of the mouse cornea with herpes simplex virus type 1 (HSV-1), the spread of virus was investigated and the types of immune cell infiltrating the trigeminal ganglion (TG) were identified in low temperature paraffin wax sections. Virus antigen was first found on day 3 and was absent after day 14. Early presentation of antigen to T cells may occur since increased expression of major histocompatibility complex (MHC) class II antigens, including de novo expression on satellite and Schwann cells, was detected in foci of such antigen on day 3. A second large peak of such expression was detected on day 10 together with increasing numbers of B and T cells. Large numbers of these lymphocytes and extensive expression of MHC class II were seen in the TG well into the phase of virus latency; the significance of this is discussed.

AIMS--This study was designed to assess the relative corneal penetration of topical drops of three antibiotics and to relate those levels to minimum inhibitory concentrations for organisms associated with bacterial keratitis. METHODS--Four drops of each of ciprofloxacin, norfloxacin, and ofloxacin (0.3% topical ophthalmic preparations) were given to 12 patients undergoing corneal transplantation. After the recipient tissue was removed, corneal drug penetration was measured using high performance liquid chromatography. RESULTS--Intracorneal concentrations of ofloxacin (geometric mean 0.81 mg kg-1) were significantly higher than both ciprofloxacin (0.60 mg kg-1; p = 0.048) and norfloxacin (0.54 mg kg-1; p = 0.012). Ciprofloxacin and norfloxacin concentrations did not differ significantly (p = 0.33). CONCLUSIONS--Review of the minimum inhibitory concentrations of the fluoroquinolones against ocular pathogens reveals that ciprofloxacin is more potent than ofloxacin against many bacteria; ofloxacin is in turn more potent than norfloxacin. These data favour the selection of ciprofloxacin and ofloxacin rather than norfloxacin for the empirical treatment of corneal infection. The greater potency of ciprofloxacin offsets the superior penetration of ofloxacin. There is a need for improved clinical trial data concerning the use of fluoroquinolone eyedrops in ulcerative keratitis; some encouraging data are available for ciprofloxacin but not (in humans) for norfloxacin or ofloxacin.

AIMS--A new sign of florid xanthelasmata is described in four male patients with orbital manifestations of Wegener's granulomatosis. METHODS--The case notes of four patients with Wegener's granulomatosis, with orbital involvement accompanied by yellow lid lesions, are reviewed. RESULTS--All the patients had active Wegener's granulomatosis at the time the lid lesions were most florid. The lesions gradually resolved as the inflammatory disease was controlled with immunosuppressive agents. The lesions displayed marked asymmetry and predominantly involved the side with the most severe inflammation. Abnormalities of lipid metabolism were not identified and it is believed that the lesions developed in a susceptible anatomical region affected by a vasculitic process. CONCLUSION--A 'yellow lid' associated with orbital inflammation and is a strong pointer to the clinical diagnosis of Wegener's granulomatosis.

We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.

Toxic effects of topical drugs may be masked by manifestations of the disease they cure. The toxicity of drug mixtures has not been thoroughly studied. We therefore investigated cytopathic effects on primary cultures of human corneal cells of six topical antimicrobials singly and in combinations of any two, to determine the combined toxicity ranking and the interaction between duration of exposure and concentration. Preconfluent cultures were exposed to fixed dilutions of single drugs, or to equal-dilution mixtures of two drugs, for 7 and 14 days. Diminishing concentrations of single drugs were applied sequentially to cultures for 14 days. The number of metabolically competent cells was assessed by measuring hexosaminidase and total protein. Toxic effects depended on substance, concentration and exposure. The scale of toxicity determined for single drugs after 7 days of exposure was: gentamicin > econazole ≥ methicillin ≥ clotrimazole ≥ miconazole ≥ chloramphenicol. After 14 days this order changed: in particular chloramphenicol showed a highly increased toxicity. The order of diminishing effects was: gentamicin > chloramphenicol ≥ methicillin > miconazole > econazole > clotrimazole. A clear reduction in cytopathic effects was observed when drug concentration was decreased progressively only in cultures treated with gentamicin or methicillin. All drug combinations were more toxic than their components at equal dilution. Combinations containing chloramphenicol ranked most toxic overall, those containing econazole least. A tapering off combination regime did not improve cell survival. These in vitro toxicity data complement clinical studies and suggest ways in which topical drugs can be chosen to minimise toxic effects to corneal surface.

Viability curves were recorded for a variety of bacteria suspended in rabbit primary aqueous humour. Controls included neat rabbit serum, dilute rabbit serum (1/50), dilute heat treated fetal bovine serum (1/50), and balanced salt solution. Rabbit aqueous humour was bactericidal in vitro for Staphylococcus aureus and Pseudomonas aeruginosa. Micrococcus spp, Streptococcus pneumoniae and Escherichia coli were not affected. The factor mediating the bactericidal effect survives filtration (0.2 micron porosity) and freezing in liquid nitrogen (-196 degrees C) but is labile when stored in air at temperatures of 4 degrees C, 25 degrees C, and 37 degrees C for 1 hour. The bactericidal effect was stable when aqueous humour was stored in an atmosphere of 5% CO2 at 37 degrees C for 3 hours. The antibacterial component in rabbit aqueous humour has yet to be identified.

Purpose: The Corneal Transplant Follow-up Study has followed 2385 corneal transplants performed in the United Kingdom and the Republic of Ireland for up to 450 days to quantify factors influencing corneal graft survival and visual outcome 3 and 12 months postoperatively. Methods: Multifactorial analyses of grafts registered by United Kingdom Transplant Support Service from July 1987 to June 1990 were used. Corrected visual acuity of functioning grafts was assessed at 3 and 12 months. Results: Of 2385 corneal transplants followed, 214 failures were observed: graft survival was 95% at 3 months and 89% at 1 year. Similar factors affected outcome at each time. Decreased risk of failure was associated with surgeons reporting most grafts, and increased risk was associated with regrafts, patients younger than 10 years of age, nonvisual reasons for grafting, endothelial failure, and deep vascularization. Visual outcome was worse in older patients and was associated with cosmetic reasons for grafting, superficial vascularization preoperatively, and secondary endothelial failure. Visual acuity was better when the other eye had been grafted previously, or when the diagnosis was keratoconus or stromal dystrophy. Conclusions: Primary endothelial failure was associated with high failure rates but good visual results when functioning. Most other factors had similar effects on both outcome measures. Improved outcome under highest-reporting surgeons was slight, and indicated possible differences in postoperative care.

Human and rabbit isolated eyes were compared, not in order to propose yet another test, but to explore the boundaries of the rabbit isolated cornea as a model for primary damage to human cornea. Changes in corneal thickness and in epithelial morphology were followed on recovery from cold-induced oedema and after exposure to the test chemicals. There was no statistical difference between control human and rabbit eyes in recovery from severe oedema. The effects of exposure to allyl alcohol, Arlagard E, NaOH (1 N), NaCl, sodium lauryl sulphate (2%) and Tween-20 were similar in human and rabbit eyes. Acetone, ethanol and glycerol had opposed-sign effects on corneal thickness in human and rabbit. From the small number of substances tested it can be concluded qualitatively that stored rabbit eyes behave in a manner similar to freshly isolated rabbit eyes, and that substances in which penetration depends on surface properties of the cornea may have species-specific effects.

Pseudotumour has rarely been reported to invade the intracranial cavity. We present a case of pseudotumour which extended intra-cranially into the posterior cranial fossa. The patient had circulating antineutrophil cytoplasmic antibodies in titres diagnostic for Wegener's granulomatosis. In the absence of clinical renal or respiratory tract disease, the case may represent a newly recognized sub-type of “localised” Wegener's granulomatosis.

Toxic effects of 10 antiviral substances [9-(2-hydroxyethoxymethyl)-guanine (ACV), E-5-(2-bromovinyl)-2′-deoxyuridine (BVDU), 5-(2-chloroethyl)-2′-deoxyuridine (CEDU), 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC), 5′-iodo-2′-deoxyuridine (IDU), phosphonoformic acid (PFA), 9-(2-phosphonylmethoxyethyl) adenine (PMEA) and 5-trifluoromethyl-2′-deoxyuridine (TFT)] on uninfected human corneal cells were evaluated in two experimental models (undisturbed adherence and growth, and wound closure) under continuous drug exposure. The antiviral drugs were ranked in order of toxicity in each of the tests. The influence on toxicity of different experimental parameters such as initial cell density, length of exposure and trauma, was evaluated. There was significant interaction between toxicity and length of exposure. In undisturbed cultures antiviral compounds ranked as follows: PMEA ≤ DHPG ≤ ACV ≤ CEDU < PFA ≤ IDU ≤ BVDU « HPMPC < TFT < HPMPA. Wounding or migration increased the apparent toxicity of the antiviral substances; trauma did not have an additional effect. In conditions where corneal epithelium is poorly populated and ulcerated, wound healing can be delayed by antiviral medication.

Areas of concern about day case surgery are highlighted. A group of 442 patients underwent cataract surgery with lens implant. They were randomly allocated to day case or inpatient groups. Questionnaires were used to assess opinions about day case cataract surgery and how patients felt they would manage. None of the areas of concern were actually a problem. Difficulties encountered by patients and clinicians in the postoperative period are discussed. Most patients appear suitable for day case surgery provided they are well informed.

The comparative cytopathic effects of a keratitis and an environmental isolate of Acanthamoeba were studied on confluent monolayers of human and rabbit corneal cells grown in culture. The presence of cells in culture induced excystment of amoebae to the active trophozoite form. Total destruction of cell monolayers was observed to be similar for both isolates, and dependent on incubation time and amoebic concentration. The relevance of these findings to human and experimental Acanthamoeba keratitis is discussed.

Herpes simplex virus (HSV) was isolated from organ cultures of anterior segments of the eyes of mice inoculated with virus on the snout or directly onto the cornea at least 5 weeks previously. The frequency of isolation of the virus was not decreased by treatment of the animals with acyclovir, suggesting that the virus is latent by the criteria usually applied. Peroxidase-antiperoxidase staining of organ cultures that had shed virus showed that viral antigens were predominantly present in the anterior uvea. Inoculation of mouse eye anterior segments in vitro showed that this tissue was the most susceptible to productive infection. These results suggest the possibility that HSV can establish a latent infection in tissues of the anterior segment of the mouse eye.

Mice were treated with serum containing antibodies to herpes simplex virus type 1 (HSV-1) or normal serum, 1 day before inoculation on the cornea with HSV-1 strain McKrae. As expected, without passive immunization, mice developed high levels of serum neutralizing antibody. By contrast, in passively immunized animals, such antibody became undetectable by 29 days after inoculation of serum, in spite of the virus infection. There was no difference between passively immunized mice and those given normal serum in the duration of shedding of virus in tears and the duration and severity of corneal epithelial disease. However, non-immunized mice had a high incidence of mortality and developed disease of the iris, corneal stroma and lids, and their corneas became opaque and vascularized. In non-immunized animals, the timing of isolation of virus from nervous tissues and the sequence of appearance of virus antigens in ocular tissues indicate that the disease of deeper eye tissue was caused by virus spreading from the nervous system back to the eye. Restriction of such spread in passively immunized animals seems the likely explanation for their protection from death and severe ocular damage. Despite this restricted spread, passively immunized animals had a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion. However, in comparison with mice given normal serum, there was a far lower incidence of such infection in the other two parts of this ganglion and in the superior cervical ganglion. Since passively immunized animals have a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion and their eyes are normal, they will prove useful in studies involving induction of recurrent disease.

During primary ocular infection of mice with herpes simplex virus type 1 (HSV-1) strain McKrae, dendritic corneal ulcers developed and many eyes became permanently damaged. When primary infection had subsided, latent infection was detected in the three parts of the trigeminal ganglion and in the superior cervical ganglion. Such latently infected mice were treated with cyclophosphamide, dexamethasone and u.v. irradiation, or cyclophosphamide and dexamethasone alone. After treatment with immunosuppressive drugs and u.v. irradiation infectious virus was isolated from the ophthalmic part of the trigeminal ganglion, and in eyelids and eyewashings; recurrent herpetic eye disease was seen but only in eyes undamaged by primary infection. After treatment with cyclophosphamide and dexamethasone alone there was a lower incidence of virus isolated from eyewashings and no recurrent disease was seen. There was a good correlation between the pattern and distribution of recurrent lesions and the distribution of cells stained due to the presence of virus antigens.

A 78-year-old female patient who had a third penetrating keratoplasty for aphakic bullous keratopathy and iris fixation of a posterior chamber intraocular lens using a single, 10-0 nylon running suture technique developed a suture track leak postoperatively. Attempts to ease the tension on the suture track reduced the astigmatism and stopped the leak temporarily. Eighteen months after the procedure, epithelial downgrowth was noted on the corneal graft on both sides of the leak site and intraocular pressure was elevated. A fourth penetrating keratoplasty combined with a trabeculectomy was performed. To our knowledge, this is the first report of epithelial downgrowth associated with suture adjustment following the single, continuous suture technique.

Aim: To investigate the development of lymphoid aggregates in the conjunctiva after corneal transplantation in rats. Methods: LEW or PVG strain corneas were transplanted orthotopically to PVG rats. Cornea and conjunctiva were examined clinically for up to 42 days. Eyes were removed with attached conjunctiva on days 10 and 15 after transplantation (before and during rejection), together with normal eyes, fixed, paraffin embedded, and examined immunohistochemically. Results: Clinically, the temporal half of the upper palpebral conjunctiva of recipients of 10/19 allografts and 1/10 isografts developed pronounced swelling, correlating with inflammation and rejection. Histologically, the swelling comprised leucocytic aggregates with an altered overlying epithelium. Aggregates contained granulocytes, macrophages, and cells expressing major histocompatibility complex (MHC) class II, CD4, and CD8, all more numerous in allograft associated conjunctiva. Class II+ cells were more abundant at the surface, whereas macrophages and T cells were more numerous in the deeper stroma. There were few B cells. There was greater CD54 expression by vascular endothelium in allograft associated aggregates. Cells expressing TNFα and IFNγ but not IL1β were present in stromal and superficial areas. Conclusions: Corneal transplantation in rats induces the development of organised conjunctival leucocytic aggregates in a fixed location that are significantly more pronounced in recipients of allografts compared with isografts and show characteristics of a Th1 type immune response. These aggregates have characteristics of conjunctiva associated lymphoid tissue and may be sites of presentation of graft antigens and lymphocyte proliferation at the ocular surface.

Herpetic stromal keratitis (HSK) and blepharoconjunctivitis in humans are thought partly to result from immunopathological responses to herpes simplex virus type 1 (HSV-1). The corneas of NIH mice were inoculated with HSV-1 (strain McKrae) and mice were examined for signs of disease and infection on days 1, 4, 7, 10, 14 and 21. The eyes and eyelids of infected and control mice were processed for immunohistochemistry and double stained for viral antigens and one of the following cell surface markers (Gr-1, F4/80, CD4, CD8, CD45R or MHC class II) or one of the following cytokines (IL-2, IL-4, IL-6, IL-10, IL-12 or IFN-γ). All infected mice developed signs of HSK by day 4 and blepharitis by day 7 and these both persisted until day 21, when signs of resolution where apparent. Virus was detected during the first week of infection and became undetectable by day 10. Large numbers of Gr-1+ cells (neutrophils) infiltrated infected corneas and eyelids in areas of viral antigen and CD4+ T cells increased significantly in number after virus clearance. In both sites, the predominant cytokines were IL-6, IL-10, IL-12 and IFN-γ, with few IL-2+ and IL-4+ cells. These observations suggest that the immune responses in the cornea are similar to those in the eyelids but, overall, the responses are not clearly characterized as either Th1 or Th2. In both sites, the neutrophil is the predominant infiltrating cell type and is a likely source of the cytokines observed and a major effector of the disease process.

To investigate clinical and histologic changes in the epithelium during corneal graft rejection in the rat.LEW (RT1(l)) or PVG (RT1(c)) strain corneas were transplanted to PVG strain recipients and examined by slit lamp for clinical signs of rejection. Recipients were killed, and corneal epithelial sheets were removed and examined by adenosine diphosphatase (ADPase) staining for Langerhans cells (LC) and by immunohistology for leukocytes and adhesion molecules (T cells, macrophages, granulocytes, major histocompatibility complex [MHC] class II, CD2 and CD54 intercellular adhesion molecule [ICAM]-1) at a range of time points before, during, and after rejection, depending on the cell type sought. Normal and contralateral eyes were examined for ADPase(+) and MHC class II(+) cells.Clinical rejection, as defined by stromal opacity, occurred between days 10 and 15 after transplantation. In 94% of allografts, a curved clinical epithelial rejection line was observed in which ADPase(+)/MHC class II(+), CD4(+), or CD8(+) T cells were identified. There were significantly more infiltrating cells of all types in epithelia of allografts than in those of isografts. The most numerous cells were CD4(+) and CD8(+) T cells, suggesting preferential migration of these cells into the epithelium from underlying layers. Expression of MHC class II and ICAM-1 was induced on epithelial cells.Epithelial rejection in rats is clinically similar to that in humans and occurs simultaneously with stromal infiltration. It may be mediated by T cells rather than macrophages. In isolation, its recognition in humans may be a useful indication that the patient is at high risk of endothelial rejection.

Background: Sorsby's fundus dystrophy (SFD) and age related macular degeneration (ARMD) are retinal diseases associated with a high level of accumulation of mutant and wild type TIMP-3, respectively, in Bruch's membrane. The pathogenic role of TIMP-3 in these diseases is uncertain, but causative mutations have been identified in the TIMP-3 gene of patients with SFD. Recent reports that TIMP-3 causes apoptosis in certain cell types and not in others prompted the authors to investigate whether TIMP-3 causes apoptosis in cultured retinal pigment epithelium (RPE) cells. Methods: RPE and MCF-7 cells (as a positive control) were initially infected with replication deficient adenovirus, to overexpress β-galactosidase (RAdLacZ) or TIMP-3 (RAdTIMP-3). TIMP-3 was detected by western blotting and ELISA. Cell viability was defined by cell counts. ISEL was used to investigate the mechanism of cell death. Results: Cultured RPE cells produced small quantities of endogenous TIMP-3 and remained viable. However, overexpression of TIMP-3 caused a dose related death of RPE cells. The mechanism of cell death was apoptosis. Conclusion: The previously unreported finding of TIMP-3 induced apoptosis of RPE cells may account for some of the early features seen in SFD and ARMD.

Purpose Early phase keratoconic comeas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. Methods Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-β. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. Results The addition of PDGF or TGF-β to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP-1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. Conclusions PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.

This study reports corneal graft survival, endothelial cell changes, and visual outcome in 20 patients who received some of the first corneas stored by organ culture in the Corneal Transplant Service Eye Bank in Bristol. Mean donor age was 48 years (SD 15, n = 20) and corneas were stored for an average of 21 days (SD 7, n = 20). Preoperative endothelial cell density was 2334 cells/mm2 (SD 235, n = 18) and this fell by 8% (SD 12) to 2158 cells/mm2 (SD 372) within the first 2 months following transplantation. In 13 patients, endothelial cell density thereafter declined exponentially with a half-life of 41 months (SD 17, n = 12; one patient excluded as an outlier). Corneas that suffered rejection episodes showed the highest rates of loss of endothelial cells. Endothelial cell loss 4 years after transplantation was 46% (SD 16, n = 12), which was similar to the postoperative decline in cell density reported for corneas stored for far shorter periods in McCarey-Kaufman medium at 4 degrees C.

Purpose: Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane. Methods: Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [3H]type I collagen. Results: The stromal tissue of fresh, normal corneas produced inactive MMP-2 of Mr 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of Mr 62,000 and Mr 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [3H]type I collagen correlated with the appearance of the Mr 43,000 activity but not the Mr 62,000 activity. Conclusion: Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.

Aims—To determine the sensitivity and specificity of culture, immunohistochemistry (IHC), the polymerase chain reaction (PCR), and in situ hybridisation (ISH) for detecting herpes simplex virus (HSV-1) in the cornea of patients undergoing penetrating keratoplasty. To compare the incidence of HSV-1 in the cornea with that of varicella zoster virus (VZV), cytomegalovirus (CMV), and EpsteinBarr virus (EBV). Methods—The corneas of 110 patients, 52 with a documented history of herpes keratitis (HSK) and 58 with non-herpetic corneal disease, were investigated using IHC, PCR, ISH, and culture. Results—HSV-1 DNA and antigen were detected in 82% and 74% respectively, of corneas of patients with HSK and in 22% and 15% of corneas of patients with no history of HSK. The sensitivity of PCR and IHC was 82% and 74% with a specificity of 78% and 85%, respectively. HSV-1 DNA and antigen were found more frequently and in increased amounts in corneas of patients with a short interval between their last attack of HSK and surgery. There was a good correlation between PCR and IHC in 71%. HSV-1 was isolated by culture in 2%. Latency associated transcripts were not detected using ISH.Evidence of VZV DNA or antigen was found significantly more frequently in the corneas of patients with a history of HSK (p

Purpose To evaluate a novel two-stage technique to increase yield of bacteria isolated from infected corneal ulcers. Methods A new blade was designed to remove friable material from infected corneal ulcers. The new blade was used in combination with standard tissue micro-homogenisation equipment in a two-stage technique intended to distribute biopsy samples evenly between relevant agar plates. Patients with presumed-bacterial corneal ulcers underwent sequential corneal sampling using the new two-stage technique and a scalpel blade, used without micro-homogenisation (the order of sampling was varied between two groups). Bacterial isolation rates were compared using the chi-squared test. Results Twenty-four patients with presumed-bacterial corneal ulcers were studied. The overall positive bacterial isolation rate was 88%, with identical bacterial isolation rates for the new two-stage technique and the scalpel blade (71%). The new technique isolated bacteria from three ulcers that had initially been ‘sterile’ when sampled with a scalpel blade. Polymicrobial infections were identified in two ulcers with the new blade where only a single organism had been identified using the scalpel blade (not significantly different). Conclusions The new two-stage technique shows promise for improving bacterial isolation rates from presumed-bacterial corneal ulcers.