[Visual recovery as the target for glaucoma]. / Visuelle Regeneration als Ziel für das Glaukom.

BACKGROUND: Visual recovery is an established but poorly studied phenomenon in glaucoma. OBJECTIVE: To provide insights into functional recovery of retinal ganglion cells (RGCs) with a view to providing information on the development of forms of treatment that improve RGC function after injury. METHOD: A model of recoverable RGC function in the mouse eye, induced by short-term elevation of intraocular pressure (IOP). RESULTS: The RGCs manifest near complete functional recovery after a prolonged period of dysfunction following acute IOP elevation. Increasing age and a high fat diet were subsequently found to impair recovery, whereas exercise substantially improved recovery such that older mice recovered in a similar way to young mice. CONCLUSION: Injured RGCs have the capacity to restore function after periods of functional impairment. Therapies that specifically target injured RGCs and enhance their capacity to recover function may provide a new approach for treating glaucoma.

Targeting retinal ganglion cell recovery.

Accumulating evidence from experimental and clinical studies suggest that retinal ganglion cells at least in the earlier stages of glaucoma have the capacity to recover function following periods of functional loss. The capacity for recovery may be negatively impacted by advancing age but can be boosted by interventions such as diet restriction and exercise.

Emerging Mitochondrial Therapeutic Targets in Optic Neuropathies.

Optic neuropathies are an important cause of blindness worldwide. The study of the most common inherited mitochondrial optic neuropathies, Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA) has highlighted a fundamental role for mitochondrial function in the survival of the affected neuron-the retinal ganglion cell. A picture is now emerging that links mitochondrial dysfunction to optic nerve disease and other neurodegenerative processes. Insights gained from the peculiar susceptibility of retinal ganglion cells to mitochondrial dysfunction are likely to inform therapeutic development for glaucoma and other common neurodegenerative diseases of aging. Despite it being a fast-evolving field of research, a lack of access to human ocular tissues and limited animal models of mitochondrial disease have prevented direct retinal ganglion cell experimentation and delayed the development of efficient therapeutic strategies to prevent vision loss. Currently, there are no approved treatments for mitochondrial disease, including optic neuropathies caused by primary or secondary mitochondrial dysfunction. Recent advances in eye research have provided important insights into the molecular mechanisms that mediate pathogenesis, and new therapeutic strategies including gene correction approaches are currently being investigated. Here, we review the general principles of mitochondrial biology relevant to retinal ganglion cell function and provide an overview of the major optic neuropathies with mitochondrial involvement, LHON and ADOA, whilst highlighting the emerging link between mitochondrial dysfunction and glaucoma. The pharmacological strategies currently being trialed to improve mitochondrial dysfunction in these optic neuropathies are discussed in addition to emerging therapeutic approaches to preserve retinal ganglion cell function.

Axonal transport along retinal ganglion cells is grossly intact during reduced function post-injury.

It has been established that beyond middle age, mice are slower to recover inner retinal function following an acute intraocular pressure (IOP) injury. While 3 month old animals exhibit near-complete recovery 1 week following injury, 12 and 18 month old animals demonstrate prolonged inner retinal dysfunction. In this study we aim to determine whether age-related differences in functional recovery of the inner retina are due to differences in retinal ganglion cell (RGC) axonal transport. C57BL/6J mice at 3 (n = 8) and 18 months (n = 8) of age were used. At day 0, right eyes were cannulated and the IOP was maintained at 50 mmHg for 30 min. At day 5, mice received bilateral intravitreal injections of choleratoxin subunit B (CTB) conjugated to Alexafluor 488. At day 7, mice were euthanized and tissue was collected. Axonal transport of CTB was quantified in retinas and superior colliculi (SC) using fluorescent microscopy. In response to IOP elevation, the overall degree of axonal transport was comparable between young and old mice. Furthermore, no differences in axonal transport were detected between control eyes and injured in mice at any age. In conclusion, impaired recovery of inner retinal function 1 week following acute IOP injury in old mice is not associated with changes in active axonal transport in RGCs at this time.

Topical prostaglandin analogues do not affect selective laser trabeculoplasty outcomes.

PURPOSE: To investigate the effect of topical prostaglandin analogue use on the efficacy of selective laser trabeculoplasty (SLT) intraocular pressure (IOP) lowering in patients with open-angle glaucoma. PATIENTS AND METHODS: This retrospective study included 123 consecutive patients who underwent 180 degrees SLT for the first time. Eyes were grouped into those that received prostaglandin analogues before and after SLT (n=74) and those that did not (n=49). The main outcome measure was IOP lowering after SLT. Success was defined as > or =20% reduction in IOP without further glaucoma intervention. RESULTS: There was no significant difference in IOP lowering at 6 months post-laser between the prostaglandin and non-prostaglandin groups (3.9+/-4.8 vs 4.6+/-3.6 mm Hg, P=0.43). Long-term SLT success rates were also not significantly different between the treatment groups (Kaplan-Meier survival analysis, P=0.68). IOP lowering at 6 months was similar in eyes that received no glaucoma medications, monotherapy with or without a prostaglandin analogue, or combination therapy with or without prostaglandin analogues (P=0.81). Logistic regression analysis showed that various patient characteristics including age, sex, type of glaucoma, previous glaucoma surgery, and other glaucoma risk factors did not predict a successful SLT outcome. However, higher pre-operative IOP was found to predict SLT success (odds ratio=1.12, 95% CI=1.02-1.24, P=0.02). CONCLUSION: The IOP lowering efficacy of SLT is not influenced by the use of topical prostaglandin analogues.

Risk factors for delayed suprachoroidal haemorrhage following glaucoma surgery.

AIM: To determine the incidence, risk factors and outcomes of delayed suprachoroidal haemorrhage (DSCH) after glaucoma surgery. METHODS: A retrospective case-control study was performed at a tertiary referral eye hospital on patients who presented with DSCH following glaucoma surgery. Cases were compared with a matched-control population that underwent equivalent procedures but did not develop DSCH. The main outcome parameters were incidence of DSCH, risk factors associated with its occurrence, visual outcome and prognostic factors. RESULTS: Of the 2752 glaucoma surgeries performed during the 10-year recruitment period, 29 cases of DSCH (1%) were identified. An increased incidence of DSCH was observed after glaucoma drainage device implantation compared with trabeculectomy-associated DSCH (p<0.0001; odds ratio 3.4; 95% CI 1.9 to 5.4). Risk factors for DSCH included low postoperative intraocular pressure (< or =3 mm Hg; p<0.001), aphakia (p<0.001), prior intraocular surgery (p<0.002), hypertension (p<0.001), anticoagulation (p = 0.002), ischaemic heart disease (p = 0.001) and respiratory disease (p = 0.008). The visual outcome of patients with haemorrhage was poor (logMAR 1.34 (SD 0.41)) and was significantly worse when compared with the control group (p = 0.002). CONCLUSIONS: In this study cohort, DSCH occurred more frequently after glaucoma drainage device implantation compared with trabeculectomy. Caution should be exercised when operating on patients with known ocular and systemic risk factors.

Trypan blue identifies antimetabolite treatment area in trabeculectomy.

AIM: Colourless solutions of mitomycin C (MMC) and 5-fluorouracil (5-FU) are widely used during trabeculectomy to inhibit postoperative scarring. The poor visibility of these agents on the eye has several drawbacks including the inability to accurately assess the area of treatment. This study examined the utility of using trypan blue dye to colour antimetabolites used during trabeculectomy and the effect of trypan blue on antimetabolite cytotoxicity in vitro. METHODS: For in vitro experiments, MMC (0.4 mg/ml) and 5-FU (25 mg/ml) were reconstituted with or without trypan blue. A lactate dehydrogenase release assay was used to measure drug induced cell death and viable cell number 7 days after treatment. For clinical assessment, trypan blue 0.1% was added to MMC and 5-FU to final concentrations of between 0.01% and 0.05%. The mixture was applied to Tenon's capsule and sclera via pre-wet or into dry 5x8 mm sponges (MMC and 5-FU) for 3 minutes or by direct subconjunctival injection after completion of surgery (5-FU). Twenty two consecutive patients undergoing trabeculectomy either with or without trypan blue were followed for 2 years postoperatively. RESULTS: The addition of 0.05% trypan blue to MMC or 5-FU did not alter MMC induced cell death or the number of viable fibroblast in vitro. In vivo, trypan blue clearly delineated the antimetabolite treatment area and facilitated control of excess antimetabolite at the wound margins as well as sponge removal. With direct subconjunctival injection, total staining area varied for a given volume with location of the needle tip. Any leakage from the injection site could be easily seen. No adverse effects attributable to trypan blue were found in 2 years of follow up. CONCLUSIONS: Trypan blue permits delineation of antimetabolite/tissue interactions without affecting cytoxicity for the assays investigated. Trypan blue can be used to visualise antimetabolite soaked sponges, estimate treatment area, and show areas of unintended tissue contact during trabeculectomy. The addition of trypan blue to antimetabolites has potential benefits in clinical, research, and teaching aspects of ocular surgery and therapy.

A pilot study of a system for grading of drainage blebs after glaucoma surgery.

PURPOSE: To develop and evaluate a novel bleb grading scheme for clinical and photographic evaluation. METHOD: A system for grading bleb photographs using widely applicable parameters was designed, and reference color photographs printed. A prospective masked agreement study was undertaken comparing slit lamp examination with mono and stereo photographs; 36 eyes of 28 patients with previous glaucoma surgery were graded according to defined parameters on a 1 to 10 scale clinically at the slit lamp by four ophthalmologists and two optometrists. Standardized stereo and mono photographs of the blebs were taken on the same day. The photographs were graded at least one week later in a masked fashion by the same observers, with grading of mono and stereo photographs also separated by one week. Analysis was performed to determine the variability and agreement between slit lamp results and photographic results, and to identify the presence of systematic bias. RESULTS: High levels of agreement were found between slit lamp and both stereo and mono photographs for vascularity indices, bleb wall thickness, and bleb elevation. Lower levels of agreement were found for the relative components of demarcated versus diffuse areas of the bleb, and for the total width of the bleb. The interquartile range for the median difference between slit lamp and photograph grading was -1.0 to 1.0 for all criteria except diffuse component (-2.0 to 2.0), and the median difference for all scores was 0.0. The median interobserver difference for all criteria was 0.0; the quartile range for all scores was between -0.5 and 1.0 except for diffuse component and width assessments whose quartiles fell in the -1.75 to 1.0 range. Examiners agreed with photographic grading within +/- 1 in more than 80% of gradings for vascularity and bleb height, within +/- 1 in more than 75% of gradings for bleb wall thickness, within +/- 2 in 61% of bleb width assessments, and +/- 2 in 59% of diffuse component. CONCLUSION: This bleb grading system is reproducible clinically and photographically. High levels of agreement between scores for photographs versus slit lamp examination were found for most categories, with good interobserver agreement for both photograph and slit lamp grading. Further refinement of scoring and reference photographs is required for optimization, especially for grading of bleb morphology.

MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule.

PURPOSE: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. METHODS: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. RESULTS: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p<0.05), and a reduction in capsule contraction was observed (p<0.05). CONCLUSIONS: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.

The effect of optic disc diameter on vertical cup to disc ratio percentiles in a population based cohort: the Blue Mountains Eye Study.

OBJECTIVE: The 97.5th percentile for vertical cup to disc ratio (VCDR) has been proposed as a useful tool to assist in the diagnosis of glaucoma in population studies. Previous reports of VCDR percentiles have either not been adjusted for disc size or have been calculated by regression analysis from small hospital based cohorts. The authors' aim was to generate VCDR percentiles in a large, population based sample. METHODS: Data were collected from 3654 individuals, aged 49 years or older, living in the Blue Mountains, west of Sydney. Vertical disc diameter and VCDR were determined by planimetry from stereo optic disc photographs. The distribution of VCDR and percentiles (95th, 97.5th, 99th) were calculated. RESULTS: 6678 eyes were included in the analysis. Median cup to disc ratio, 95th, 97.5th, and 99th percentile increased with vertical optic disc diameter in a linear fashion. An increase of 0.2 in median VCDR (0.35 to 0.55) was observed between small (1.1-1.3 mm) and large (1.8-2.0 mm) optic discs. An equivalent increase of 0.2 (0.59 to 0.74) was observed for the 97.5th percentile from small to large discs. CONCLUSION: VCDR percentiles for a "normal" population, adjusted for vertical optic disc diameter are presented. One quarter of all discs fell within the small or large disc categories highlighting the importance for estimating optic disc size. These data may assist in the diagnosis of glaucoma in clinical practice as well as providing a normative database. Sole use of VCDR percentile cut offs in defining glaucoma cases in population surveys requires further validation.

Evaluating clinical signs in trabeculectomized eyes.

AIM: To evaluate interobserver agreement for clinical signs in trabeculectomized eyes when examined face-to-face with slit-lamp biomicroscopy (SL) or by remote examination using telemedicine (real-time remote video imaging; TM). METHOD: A system for examining trabeculectomized eyes was devised and validated. A prospective randomized interobserver agreement study was then undertaken to compare standard SL biomicroscopy and TM. Remote examination was performed using a 384 kbps Sony 5100 videoconferencing system. Three ophthalmologists each examined 40 eyes of 40 patients, who had previously undergone trabeculectomy. In rotation, two examiners used SL biomicroscopy. The third examined the eye remotely by TM. Analysis was performed to determine the variability in clinical signs and the presence or absence of systematic bias between ophthalmologists and examination methods. RESULTS: High levels of agreement were observed for paired examinations by SL biomicroscopy (SL/SL) for bleb vascularity (score range 0-10) with no systematic bias. Paired examination by SL and TM (SL/TM) also showed good levels of agreement for bleb vascularity, although the spread of disagreement was wider (95% limits of agreement 2.57 vs 2.98 (P=0.054)). For anterior chamber depth, observers agreed within +/- 10% of anterior chamber depth for 68% of eyes (SL/SL) and 51% of eyes (SL/TM) (P=0.68). Agreement was 'good' for wall thickness (kappa=0.63 +/- 0.08), bleb height (kappa=0.67 +/- 0.1), and the existence of bleb leak (kappa=0.63 +/- 0.19), but poor for bleb morphology (kappa=0.26 +/- 0.12). For the SL/TM comparison, agreement was fair for wall thickness (kappa=0.39 +/- 0.13), poor for bleb height (kappa=0.17 +/- 0.12), good for bleb leak (kappa=0.56 +/- 0.19), and fair for bleb morphology (kappa=0.31 +/- 0.12). Microcysts were not reliably detected using either technique. CONCLUSION: SL biomicroscopy and TM telemedicine examination may permit reliable clinical assessment of trabeculectomized eyes. However, remote examination with TM is more limited with respect to assessing bleb height and bleb wall thickness. The assessment of bleb morphology and microcysts was unreliable with both instruments. We propose that TM examination of trabeculectomized eyes appears safe and appropriate in situations where face-to-face examination by an ophthalmologist is not practical.

T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts.

AIMS: To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon's capsule fibroblasts. METHODS: IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. RESULTS: T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). CONCLUSION: T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically.

The effects of single doses of beta radiation on the wound healing behaviour of human Tenon's capsule fibroblasts.

AIM: To determine the effects of single doses of beta radiation on the wound healing functions of human Tenon's capsule fibroblasts (hTf). METHODS: hTf were grown in tissue culture and irradiated with beta radiation using a strontium 90 source. The effects of beta radiation on fibroblast migration was studied using microporous transwell membranes. The effects of radiation on fibroblast contraction was investigated using a fibroblast populated collagen gels model. Production of extracellular matrix molecules (collagen I, collagen III, and fibronectin) by monolayers of irradiated fibroblasts was quantified for 14 days following single doses of beta radiation. RESULTS: Growth inhibiting doses of beta radiation did not inhibit fibroblast migration or contraction at any time point. Levels of soluble fibronectin from irradiated populations were significantly reduced after >500 cGy beta radiation. Collagen I and III levels were not reduced after any dose of radiation, and increased following treatment with 1000 cGy beta radiation. CONCLUSIONS: Growth arresting doses of beta radiation have unique effects on the wound healing behaviour of human Tenon's capsule fibroblasts. There was no significant effect on cellular migration or contraction, but ECM production was altered. Fibronectin production was inhibited following higher radiation doses, and collagen I and III production increased after 1000 cGy. The effects of single doses of beta radiation on ocular fibroblast wound healing behaviour are very different from those of 5-fluorouracil and mitomycin C, and these differences may be exploited clinically in the regulation of wound healing after glaucoma filtration surgery.

In vivo production of interferon beta by human Tenon's fibroblasts; a possible mediator for the development of chronic conjunctival inflammation.

BACKGROUND: Chronic inflammation may develop from failure of the immune system to deactivate itself during resolution of the wound healing response, and is recognised as a major risk factor for trabeculectomy failure. Fibroblast/T cell interactions may contribute to aggressive scarring. Our previous research showed that in vitro human Tenon's fibroblast produced interferon beta was responsible for preventing T cell apoptosis, suggesting that this interaction could contribute to the development of chronic inflammation. METHODS: Immunohistological techniques were used to investigate the in vivo components of this particular fibroblast/T cell interaction in conjunctival biopsies from glaucoma patients undergoing filtration surgery. RESULTS: Fibroblast produced interferon beta and T lymphocytes were identified in human conjunctiva. CONCLUSION: The components of fibroblast mediated prevention of T cell apoptosis were identified in vivo, suggesting that the development of this interaction is possible and that it may contribute to the development of chronic inflammation and excessive scarring.

Human tenon's fibroblast-produced ifnbeta and the prevention of t-cell apoptosis.

PURPOSE: Fibroblast-T-cell interactions may contribute to the development of chronic inflammation, a risk factor for trabeculectomy failure. This study was undertaken to determine whether normal and growth-arrested human Tenon's fibroblasts (HTF) can prevent cytokine deprivation-mediated T-cell apoptosis through the secretion of interferon (IFN)beta. METHODS: HTF were used either untreated or pretreated with mitomycin-C (MMC; 0.1 or 0.4 mg/ml) or 5-fluorouracil (5FU; 25 or 50 mg/ml). IL2-deprived T cells were cocultured with HTF. T-cell viability was measured at specific time points. Human Tenon's fibroblast-conditioned medium was used either untreated or treated with a neutralizing antibody against IFNbeta to block its action, after which IL2-deprived T cells were added and T-cell viability was measured. An image analysis system was used to determine the production of IFNbeta by either untreated or MMC-treated HTF. RESULTS: T-cell viability was significantly greater when T cells were cocultured with both untreated and growth-arrested HTF than when T cells were cultured alone (day 7, P = 0.0001). Neutralizing the action of IFNbeta blocked HTF-mediated T-cell rescue from apoptosis. Both untreated and growth-arrested HTF secrete IFNbeta, and MMC at 0.4 mg/ml appeared to increase IFNbeta production. CONCLUSIONS: Cytokine deprivation-mediated T-cell apoptosis can be prevented by the action of IFNbeta secreted by both normal and growth-arrested HTF, which suggests that growth-arrested HTF can still participate in an aggressive wound-healing reaction by mediating a persistent inflammatory phase. This may partly explain why some trabeculectomies fail in high-risk patients, despite the use of antimetabolites.

An optometrist's role of co-management in a hospital glaucoma clinic.

In recent years, optometrists have started to work in some clinical roles in hospital medical clinics. This paper outlines the current working practice in a typical glaucoma clinic at Moorfields Eye Hospital NHS Trust and goes on to establish the ability of an optometrist working with a team of ophthalmologists. In this study, 54 patients (n = 108 eyes) were recruited and clinically assessed by an optometrist. Subsequently, a research fellow (i.e. ophthalmologist) assessed the same patients independently. The results were compared in order to determine the accuracy of the optometrist's evaluation and proposed management. This paper considers the costs of employing personnel by reviewing the salaries of optometrists and medical assistants. Although nurses have started to work alongside ophthalmologists, and orthoptists may be considering such work, it is not within the scope of this paper to consider either of these groups. If optometrists are to become an accepted part of a glaucoma clinical team, consideration needs to be given to the training and experience they receive.

The role of the immune system in conjunctival wound healing after glaucoma surgery.

The immune system has a fundamental role in the development and regulation of ocular healing, which plays an important role in the pathogenesis of most blinding diseases. This review discusses the mechanisms of normal wound healing, describing the animal and fetal wound healing models used to provide further insight into normal wound repair. In particular, conjunctival wound repair after glaucoma filtration surgery will be used to illustrate the contributions that the different components of the immune system make to the healing process. The potential role of macrophages, the possible regulatory effect of lymphocytes, and the important role of growth factors and cytokines in the wound healing reaction are discussed. The significance of the immune system in the pathogenesis of aggressive conjunctival scarring is addressed, particularly assessing the predisposing factors, including drugs, age, and ethnicity. The rationale behind the pharmacological agents currently used to modulate the wound healing response and the effects these drugs have on the function of the immune system are described. Finally, potential new therapeutic approaches to regulating the wound healing response are reported.

Effects of antimetabolite induced cellular growth arrest on fibroblast-fibroblast interactions.

The use of single five-minute applications of antimetabolites during glaucoma filtration surgery has significantly reduced the occurrence of post-operative scarring and bleb failure. However, surgery for some patients is still unsuccessful, despite the use of antiproliferative agents, due to formation of scar tissue at the drainage site. It is not known if cells growth arrested in the treated area with a single application of antimetabolites influence the activity of adjacent non-treated cells. We hypothesise that the activity of non-treated cells recruited to the wound site may be involved in post-operative scarring. The aim of this study was to investigate the effects of antimetabolite induced cellular growth arrest on cell-cell interactions using in vitro techniques.Tenon's capsule fibroblast cultures were growth arrested by exposure for 5 minutes to mitomycin-C (0.001, 0.01 and 0.1 mg ml-1), 5-fluorouracil (0.25, 2.5 and 25 mg ml-1) or phosphate buffered saline (PBS). Following a period of serum-starvation, conditioned media (CM) were subsequently collected from the cells at intervals up to 29 days post-treatment. Correction for cell number was made prior to supplementation of serum-free medium with CM. CM were assessed for ability to support or inhibit normal non-treated fibroblast proliferation, migration and collagen contraction. Conditioned media collected from cells growth arrested with MMC or 5FU stimulated normal fibroblast proliferation, migration and collagen contraction in excess of non-conditioned serum-free medium. Peaks of fibroblast activity in CM differed according to which drug and concentration had originally been given to the treated cells. This study has demonstrated that CM collected from fibroblasts treated for 5 minutes with a range of concentrations of antimetabolites can differentially influence normal non-treated fibroblast activity. This in vitro data suggests that despite entering growth arrest, fibroblasts may still influence the behaviour of other cells via soluble mediators. They may have implications in the clinical setting, in that it may not be sufficient to suppress proliferation alone to prevent fibroblast behaviour associated with scarring after glaucoma filtration surgery.