MD Felix Treumer
email: ftreumer@auge.uni-kiel.de

MD Benjamin Sattler

MD Arnd Bunse

PhD Alexa Klettner

MD Jost Hillenkamp

MD Johann Roider

Experimental microablation of choroidal tissue for autologous retinal pigmentepithelium-choroid translocation with the pulsed electron avalanche knife (PEAK-fc)

Keywords: RPE transplantation, pulsed electron avalanche knife PEAK-fc, choroidal ablation, AMD

Aim: To test the microablation of excess graft choroidal tissue with the pulsed electron avalanche knife (PEAK-fc) in an in-vitro model of autologous retinal pigment epithelium (RPE)-choroid translocation.

Methods: Choroidal tissue of porcine RPE-choroid explants was ablated with the PEAK-fc. Tissue morphology was assessed by light microscopy (LM) and scanning electron microscopy (SEM). The amount of ablated choroidal tissue was analysed as a function of three PEAK-fc parameters: (1) amplitude of biphasic voltage (70-100%), (2) distance between choroidal tissue and tip of the PEAK-fc (0-300 µm), (3) exposure time (2-8 s). Results: LM and SEM showed a smooth plain within the ablation area with well defined cutting edges and preserved adjacent tissue structure. The mean amount of ablated tissue correlated linearly with (1) applied voltage (range: 79 to 120 µm, r=0.34) and (2) distance between choroidal tissue and PEAK-fc tip (range: 10 to 100 µm, r=0.74). (3) The mean amount of ablated tissue increased with exposure time between 2 and 4 s (36 to 88 µm, r=0.4) and remained constant between 4 and 8 s. Conclusion: The PEAK-fc accurately microablates choroidal tissue in-vitro. The adjacent choroidal tissue structure and Bruch`s membrane are preserved. Patient studies are required to test the PEAK-fc in RPE-choroid translocation surgery.

Introduction

The currently most widely applied treatment of exudative age related macular degeneration is based on anti-vascular endothelia growth factor (VEGF) agents. However, in certain cases of neovascular AMD treatment with anti-VEGF agents cannot be applied, e.g. massive subretinal hemorrhage or tears of the retinal pigment epithelium (RPE). Affected patients suffer severe loss of vision due to scarring of the subretinal tissue or degeneration of the photoreceptors secondary to the lack of support of an intact subfoveal RPE layer. One possibility to restore a functioning RPE layer is the surgical excision of the underlying choroidal neovascular membrane (CNV) followed by translocation of an autologous peripheral RPE-choroid patch to the subfoveal area. [START_REF] Binder | Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial[END_REF][START_REF] Maclaren | Autologous transplantation of the retinal pigment epithelium and choroid in the treatment of neovascular age-related macular degeneration[END_REF][START_REF] Van Meurs | Autologous retinal pigment epithelium and choroid translocation in patients with exudative age-related macular degeneration: short-term follow-up[END_REF][START_REF] Heussen | Autologous translocation of the choroid and RPE in age-related macular degeneration: 1-year follow-up in 30 patients and recommendations for patient selection[END_REF][START_REF] Treumer | Autologous retinal pigment epithelium-choroid sheet transplantation in age related macular degeneration: morphological and functional results[END_REF][START_REF] Maaijwee | Retinal pigment epithelium (RPE)-choroid graft translocation in the treatment of an RPE tear: preliminary results[END_REF] Although retinal function was detected over these grafts visual outcome often remained unsatisfactory and the long-term survival of the graft is often limited. [START_REF] Maclaren | Long-term results of submacular surgery combined with macular translocation of the retinal pigment epithelium in neovascular age-related macular degeneration[END_REF] This may in part be due to a co-translocation of large amounts of choroidal tissue together with Bruch's membrane and the RPE. As conventional RPE-choroid grafts contain more than 90% choroidal tissue in vertical dimension, the graft choroid may be expected to increase the path length of diffusion of oxygen and nutrients between what little is left of functionally intact macular choroid and the overlying outer retina. [START_REF] Ramrattan | Morphometric analysis of Bruch's membrane, the choriocapillaris, and the choroid in aging[END_REF] However, the underlying premise of excess graft choroid as a barrier to the diffusion of nutrients is a reasonable hypothesis but the hypothesis has so far not been experimentally tested. Connections between macular and graft choroidal vessels with resulting graft perfusion have been demonstrated by angiography studies. [START_REF] Maaijwee | Angiographic evidence for revascularization of an rpe-choroid graft in patients with age-related macular degeneration[END_REF] A second hypothesis is that in cases with insufficient revascularisation, fibrosing graft choroidal tissue may form a diffusion barrier between functionally intact choroid and translocated RPE resulting in graft failure and loss of visual function. The pulsed electron avalanche knife (PEAK-fc) is an electrosurgical device suitable for intraoperative tractionless preparation of intraocular structures such as epiretinal membranes, e.g. in proliferative vitreoretinopathy or proliferative diabetic retinopathy. [START_REF] Palanker | Pulsed electron avalanche knife (PEAK) for intraocular surgery[END_REF][START_REF] Palanker | Effects of the pulsed electron avalanche knife on retinal tissue[END_REF][START_REF] Priglinger | Pulsed electron avalanche knife (PEAK-fc) for dissection of retinal tissue[END_REF] The PEAK-fc works by the induction of plasma in conductive medium generated by microsecond pulses of a high electric field leading to a dissection and ablation of tissue at close proximity of the 50 µm wire microelectrode. As the pulses do not exceed 100 µs in duration the heat diffuses to the surrounding tissue only up to 7 µm inducing only little collateral thermal damage. [START_REF] Priglinger | Pulsed electron avalanche knife: new technology for cataract surgery[END_REF] The aim of this study was to investigate in-vitro efficacy and safety of tissue ablation with the PEAK-fc knife to reduce the amount of graft choroidal tissue in RPE-choroid translocation surgery.

Materials and methods

Tissue ablation

The anterior segment and vitreous of freshly enucleated porcine eyes was removed. The sclera was carefully dissected from the RPE-choroid layer using microsurgical scissors and forceps. The retina was removed and the RPE-choroid explant was pinned with the RPE facing down on a microfibre cloth and was then fixed to a cork board in 0,9 % sodium chloride solution.The tissue was viewed under a surgical microscope and the PEAK-fc (Carl Zeiss Meditec, Jena, Germany) was fixed in a micromanipulator to precisely adjust the distance between the surface of the choroidal tissue and the PEAK probe during the ablation process. To identify the treated areas in the histological cross sections, these regions were marked with a marker pen (WAK-Chemistry, Bad Homburg, Germany). To simulate the intraoperative situation choroidal tissue was ablated handheld with smooth, sweeping movements in a second set of experiments.

We analysed the thickness of the ablated choroidal tissue (µm) as a function of the following PEAK-fc parameters: (1) amplitude of the biphasic voltage (displayed as percent of the maximum value of 600 V), (2) exposure time (2 -8 s), and (3) distance between the surface of the choroidal tissue and the tip of the PEAK-fc (0 -300 µm). In preliminary ablation experiments using porcine choroidal tissue we established standard values for the following parameters: voltage 90%, exposure time 8 seconds, distance 100 µm, repetition rate 100 Hz, and 20 pulses per burst. In the present study we altered either voltage, exposure time, or distance each experiment while the other four parameters were set to standard. Each parameter was tested 6 to 29 times. The same investigator ablated the tissue and measured ablation depth in the histological cross sections (BS).

Visual control of the ablation

To evaluate if the depth of the ablation can be visually assessed we developed a visual score system: 0 = no ablation assumed, 1 = change of tissue colour, 2 = light ablation, 3 = medium ablation, 4 = strong ablation assumed. Using this score system we estimated the depth of the ablation during the ablation procedure and compared these estimated values to the measured ablation depth in the paraffin sections.

Light microscopy (LM) and scanning electron microscopy (SEM)

For LM the RPE-choroid explants were fixed in 4% formaldehyde, dehydrated in a graded series of ethanol and embedded in paraffin. 3 µm sections were stained with hematoxylineosin and examined with light microscopy. Digital images were obtained with the AxioCamMRc5 (Zeiss, Jena, Germany) and the thickness of the ablated choroidal tissue was analysed by manual positioning of markers at the bottom of the ablation and at the edge of the ablation using the software AxioVisionAC, version 4.1 (Zeiss, Jena, Germany).

For SEM the tissue was fixed for 24 hours in 3% glutaraldehyde and rinsed with phosphate buffered saline over night. The tissue was postfixed in 1% osmium tetroxide for 90 min and dehydrated in a graded series of ethanol. The tissue was dried with CO 2 in the Critical Point Dryer (Blazer, Lichtenstein) and sputter-coated with gold followed by digital image acquisition.

Statistical methods

In the statistical analysis the type I error (alpha) was set at 0.05. The correlation between voltage (% of maximum), exposure time (s), and distance (µm ) and the thickness of ablated choroidal tissue (µm) was assessed with the Spearman rho and Kendall tau b correlation coefficients. The results were statistically analysed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA).

Results

Tissue ablation

Parameter "voltage":

The mean amount of ablated tissue increased linearly with the applied voltage. The application of 70% of the maximal voltage (n=10) produced a mean ablation of 79 µm (SD ±27). 80% of voltage (n=11) lead to a mean ablation of 98 µm (SD ±16), 90% of voltage (n=29) resulted in a mean ablation of 111 µm (SD ±48) and maximal voltage (100%, n=10) produced a mean ablation of 120 µm (SD ±24). The correlation coefficient Spearman rho was 0.34, p=0.007, Kendall tau was 0.26, p=0.01 (Fig. 1).

Parameter "distance":

The greatest amount of choroidal tissue was ablated when the tip had direct contact to the choroidal tissue (mean ablation 100 µm (SD ±49), n=29). The amount of ablated tissue decreased linearly to 65 µm (SD ±25) at a distance of 100 µm (n=9), to 49 µm (SD ±33) at a distance of 200 µm (n=8) and to 9 µm (SD ±11) ablation depth at a distance of 300 µm (n=9). Spearman rho was calculated 0.74, p=0.01, Kendall tau was 0.6, p=0.01 (Fig. 2).

Parameter "exposure time":

Increasing the exposure time from 2 (n=6) to 4 (n=11) seconds increased the mean tissue ablation from 36 µm (SD ±15) to 88 µm (SD ±33). Spearman rho 0.4, p=0.01, Kendall tau 0.3, p=0.01. Longer exposure time (6 s (n=10) and 8 s (n=19)) did not result in a significant increase of tissue ablation (Fig. 3).

Visual control of the ablation

The visually estimated ablation depth correlated linearly with the ablation depth determined by histology (Spearman rho = 0.69, p=0.01). When no tissue ablation (score = 0) was estimated, the mean histological ablation was 10 µm (SD ±11), score 1 revealed mean ablation of 38 µm (SD ±27), score 2 corresponded to mean ablation of 62 µm (SD ±25), score 3 corresponded to 91 µm (SD ±35) ablation and score 4 revealed 120 µm (SD ±41) mean ablation depth in the histological cross sections (Fig. 4).

LM and SEM

Both LM and SEM showed sharp cutting edges and preserved adjacent tissue structure. Tissue ablation resulted in a smooth plain within the entire ablation area (Fig. 5).

To simulate the intraoperative situation we performed tissue ablation in a second set of experiments with the PEAK-fc handheld. A smooth ablation was achieved with gentle, sweeping movements of the probe wire of the PEAK-fc over the choroidal surface of the explants. We found that careful bending of the probe wire prevented entanglement of the wire with the choroidal loose connective tissue.

Discussion

The results of this study show that the PEAK-fc accurately ablates choroidal tissue of porcine RPE-choroid explants. SEM revealed well demarcated areas of ablation and a smooth plain within the entire ablation area. Smooth ablation was achieved when the PEAK-fc was fixed and also when it was moved over the choroidal surface of the explants handheld with gentle, sweeping movements. Significant thermal collateral damage was neither detected by LM nor by SEM. We did not perform in-vivo experiments but we used freshly enucleated porcine eyes with a post-mortem time of less than 6 hours. Therefore, the histologic findings are likely to be similar to the in-vivo situation. The amount of ablated tissue increased linearly with the applied voltage and decreased linearly with the distance between the tip of the knife and the tissue. The visually estimated amount of ablated tissue correlated well with LM histological measurements. The PEAK-fc works by the induction of plasma close to the microelectrode. Theoretically, extended exposure time cannot result in damage to tissue further distant from the electrode. Indeed, we found that the amount of ablated tissue increased with the exposure time between 2 and 4 s but remained unchanged when the exposure time was further extended. Intraoperatively, this mechanism could prevent inadvertent damage to deeper tissue layers such as Bruch`s membrane.

Hu and Zhang et al. abraded graft choroidal tissue in an in-vivo rabbit model with a sharp spatula. They found intact retina on debrided grafts indicating good functional support of the overlying retina. [START_REF] Hu | Autologous transplantation of RPE with partial-thickness choroid after mechanical debridement of Bruch membrane in the rabbit[END_REF] Further experiments confirmed that partial thickness RPE-choroid grafts support photoreceptor cell survival and also initiate early repair mechanisms as exhibited by photoreceptor outer segment regeneration. [START_REF] Zhang | Photoreceptors repair by autologous transplantation of retinal pigment epithelium and partial-thickness choroid graft in rabbits[END_REF] However, to abrade the choroidal tissue with a spatula is a comparatively rough procedure with limited accuracy exposing the graft to tractional forces and mechanical stress. The PEAK-fc allows gentler and more controlled ablation of choroidal tissue avoiding mechanical stress with traction and distortion.

Intraoperatively, the PEAK-fc could possibly also be used to cut out the RPE-choroid graft.

The device has previously been shown to produce effective and reproducible cuts of retinal tissue. [START_REF] Priglinger | Pulsed electron avalanche knife (PEAK-fc) for dissection of retinal tissue[END_REF] In accordance with our findings the PEAK-fc produced sharp edges and collateral coagulation damage could be reduced to minimal changes using pulse parameters within a range of 350-380 V, pulse repetition rate 300 Hz, pulse duration 100 µs, and 30 pulses per burst.

The PEAK-fc for ablation of choroidal tissue could be further improved. We found that careful bending of the probe wire prevented entanglement of the wire with the choroidal loose connective tissue. To address this problem a modification of the plasma knife with a bladelike electrode could be helpful: Palanker et al. developed a 12 µm thin foil electrode with insulated sides and an exposed edge. Flat sides of the electrode are coated with glass, leaving only the semi-cylindrical edge of the foil with a radius of curvature of 6 µm exposed to the conductive physiological medium. Dimensions of the blade electrodes can be adjusted for different applications, e.g. for intraocular use 1 mm in length and 0.6 mm in width. [START_REF] Palanker | Electrosurgery with cellular precision[END_REF] This semi-cylindrical shape may facilitate the intraoperative ablation of choroidal tissue.

During surgery the PEAK-fc knife has to be used inside the eye because removal of the graft for ablation would most likely lead to loss of RPE-cells and include a risk of graft infection. In this regard a potential drawback of the PEAK-fc is the formation of small gas bubbles during the operation. However, Priglinger et al. used the PEAK-fc to cut retina during vitrectomy and the amount of gas bubble formation was comparable to conventional intraocular diathermy [13;17] In none of the performed procedures the development of gas impaired vision to such a degree as to render surgery unsafe or impossible.

As an alternative method of tissue thickness reduction of RPE-choroid patches Holz et al.

tested a 308 nm UV-B excimer laser. The laser cleanly and accurately separated choroidal tissue borders without thermal collateral damage in human donor eyes. However, due to the interindividual differences in tissue properties, a broad spectrum of laser energies had to be applied. [START_REF] Holz | Intraocular microablation of choroidal tissue by a 308 nm AIDA excimer laser for RPE-transplantation in patients with agerelated macular degeneration[END_REF] Furthermore, the method may be obsolete for clinical application because the laser induced apoptosis in human ARPE-19 cells at energy levels below the choroidal ablation threshold. [START_REF] Krohne | Effect of 308 nm excimer laser irradiation on retinal pigment epithelium cell viability in vitro[END_REF] In summary, the PEAK-fc accurately and reproducibly microablates choroidal tissue without significant collateral damage of adjacent tissue. The ablation depth is well controlled and extended exposure times do not lead to inadvertent damage of deeper tissue layers such as Bruch`s membrane. The PEAK-fc knife is a prototype. To our knowledge, the results of the present investigation and the cited work of others represent the currently available data on safety. As this experimental data is preliminary, extra caution should be applied when using the PEAK-fc knife in RPE-choroid patch translocation surgery. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing group Ltd to permit this article (if accepted) to be published in BJO and any other BMJPGL products and sublicenses such use and exploit all subsidiary rights, as set out in our licence.

Legends

Competing interest: None declared.

Figure 1 .

 1 Figure 1. Mean (±SD) amount of ablated choroidal tissue (µm) as a function of the PEAK-fc parameter "voltage" (displayed as percent of maximum values).

Figure 2 .

 2 Figure 2. Mean (±SD) amount of ablated choroidal tissue (µm) in relation to the PEAK-fc parameter "distance" of the PEAK-fc tip from the tissue surface (0-300 µm).

Figure 3 .

 3 Figure 3. Mean (±SD) amount of ablated choroidal tissue (µm) in relation to the PEAK-fc parameter "exposure time" (2 to 8 seconds).

Figure 4 .

 4 Figure 4. Correlation of visually estimated choroidal ablation depth (score 0-4) with the ablation depth (µm) determined by histology.

Figure 5 .

 5 Figure 5. a) Photomicrograph of hematoxylin-eosin stained section of choroidal tissue ablated with the PEAK-fc (voltage 90%, distance 0 µm, time: 8 s). The adjacent tissue appears well preserved. b, c) Photomicrographs of scanning electron microscope sections of ablations with different parameters of the PEAK-fc: b) voltage: 90%, distance: 100 µm, time: 2 s. The ablation area is well demarcated with shallow and constant deepening without irregularities. c) voltage: 90%, distance: 0 µm, time: 8 s. The ablation is deeper as compared to b) with a smooth ablation surface and well preserved adjacent tissue.

Figure 5

 5 Figure 1


Heather C Russell
email: heatherrussell74@googlemail.com

Vikas Chadha

David Lockington

Ewan G Kemp

Topical mitomycin C chemotherapy in the management of ocular surface neoplasia: a 10 year review of treatment outcomes and complications

Introduction

Use of topical mitomycin C (MMC) has gained popularity in the management of ocular surface neoplasia. The aim of this study was to determine outcomes and complications following such treatment.

Methods

This was a retrospective review of patients treated with topical MMC for ocular surface neoplasia including primary acquired melanosis (PAM), melanoma, corneal-conjunctival intraepithelial neoplasia (CCIN), squamous cell carcinoma (SCC), and sebaceous gland carcinoma (SGC). Data regarding diagnosis, short and long-term outcomes, and short and long-term complications were recorded.

Results

Fifty-eight patients were identified, with a mean age of 63 years and mean follow-up of 36 months. Twenty-one received MMC as primary therapy, and 37 as surgical adjuvant. The regimen was 0.04% MMC QID three weeks on, three weeks off, three weeks on, with topical steroid and lubricants throughout. Initial clinical response was either partial or complete in 93%. Overall, 26% developed recurrent disease at a mean of 13 months post treatment.

Recurrence rates by pathology were PAM 20%, melanoma 25%, CCIN 0%, SCC 67% and SGC 57%. Short-term complications occurred in 52%, but only 7% required treatment cessation.

Long-term complications occurred in 31%; commonly continuing keratoconjunctivitis, epiphora and corneal problems.

Conclusion

The results confirm the effectiveness of topical MMC chemotherapy in the management of ocular surface neoplasia. Self-limiting short-term complications were common, however limbal stem cell deficiency appears to be a significant long-term complication of treatment, occurring in 12%.

INTRODUCTION

Mitomycin C (MMC) is an antibiotic isolated from Streptomyces caespitosus. It undergoes metabolic activation to become an alkylating agent that is cytotoxic to both proliferating and non-proliferating cells. [START_REF] Abraham | Mitomycin: Clinical Applications in Ophthalmic Practice[END_REF] Over the past 16 years topical use of MMC has gained popularity in the management of ocular surface neoplasia, with the 'whole eye treatment' offering potential benefits over excision and cryotherapy for the treatment of extensive and subclinical disease.

Primary acquired melanosis is a superficial patchy pigmentation of the conjunctiva which can lead to conjunctival melanoma in up to 30% of affected patients. [START_REF] Shields | Dramatic regression of conjunctival and corneal acquired melanosis with topical mitomycin C[END_REF] Topical MMC chemotherapy as a primary treatment has been found to be very effective in reducing and even eliminating pigmentation, with recurrence rates ranging from 13 -38%. [START_REF] Shields | Dramatic regression of conjunctival and corneal acquired melanosis with topical mitomycin C[END_REF][START_REF] Pe'er | The treatment of primary acquired melanosis (PAM) with atypia by topical mitomycin C[END_REF][START_REF] Demirci | Topical mitomycin chemotherapy for conjunctival malignant melanoma and primary acquired melanosis with atypia: clinical experience with histopathologic observations[END_REF] The role of topical MMC in the management of conjunctival melanoma is however less clear, with reported recurrence rates of 33 -100% for primary treatment, [START_REF] Pe'er | The treatment of primary acquired melanosis (PAM) with atypia by topical mitomycin C[END_REF][START_REF] Demirci | Topical mitomycin chemotherapy for conjunctival malignant melanoma and primary acquired melanosis with atypia: clinical experience with histopathologic observations[END_REF][5] and 0-66% for adjuvant treatment. [START_REF] Demirci | Topical mitomycin chemotherapy for conjunctival malignant melanoma and primary acquired melanosis with atypia: clinical experience with histopathologic observations[END_REF][5][START_REF] Shields | Management of conjunctival and corneal melanoma with surgical excision, amniotic membrane allograft, and topical chemotherapy[END_REF] Ocular surface squamous neoplasia (OSSN) is a rare condition with a range of clinical presentations from intraepithial involvement with no frank invasion -corneal-conjunctival intraepithelial neoplasia (CCIN), through to invasive sqamous cell carcinoma (SCC). [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] Following the first report of use of topical MMC in the treatment of CCIN, [START_REF] Fucht-Pery | Mitomycin C therapy for corneal intraepithelial neoplasia[END_REF] many other studies have confirmed its effectiveness as both a primary and adjuvant therapy, with very low recurrence rates of 0 -22%. [9][START_REF] Chen | Mitomycin C as an adjunct in the treatment of localised ocular surface squamous neoplasia[END_REF][11][START_REF] Rozenman | Treatment of conjunctival intraepithelial neoplasia with topical drops of mitomycin C[END_REF] It is also effective in the management of minimally elevated SCC, with recurrence rates as low as zero, [START_REF] Frucht-Pery | Topical mitomycin-C for partially excised conjunctival squamous cell carcinoma[END_REF][14] and as a neoadjuvant 'chemoreductant' followed by surgical excision for thicker tumours. [START_REF] Shields | Chemoreduction with topical mitomycin C prior to resection of extensive squamous cell carcinoma of the conjunctiva[END_REF] Sebaceous gland carcinoma (SGC) is a rare tumour most commonly affecting the upper lid.

Due to its propensity to pagetoid spread within the conjunctiva, management of this tumour is notoriously difficult, often necessitating exenteration. [START_REF] Chao | Outcome of patients with periocular sebaceous gland carcinoma with and without conjunctival intraepithelial invasion[END_REF] Favourable results have been reported with use of topical MMC as an adjuvant in the management of SGC with pagetoid spread. [START_REF] Shields | Topical mitomycin-C for pagetoid invasion of the conjunctiva by eyelid sebaceous gland carcinoma[END_REF][START_REF] Tumuluri | Mitomycin C in sebaceous gland carcinoma with pagetoid spread[END_REF][START_REF] Rudkin | Mitomycin-C as adjuvant therapy in the treatment of sebaceous gland carcinoma in high-risk locations[END_REF] Reported complications of topical MMC chemotherapy range from common transient selflimiting problems such as local irritation, hyperaemia, epiphora, allergy, keratoconjunctivitis and corneal abrasion, [START_REF] Fucht-Pery | Mitomycin C therapy for corneal intraepithelial neoplasia[END_REF]14,[START_REF] Shields | Topical mitomycin-C for pagetoid invasion of the conjunctiva by eyelid sebaceous gland carcinoma[END_REF]20] to longer lasting problems such as punctal stenosis, cataract, persisting keratoconjunctivitis, and limbal stem cell deficiency. [20][START_REF] Sacu | Intumescent cataract after topical mitomycin C for conjunctival malignant melanoma[END_REF][START_REF] Dudney | Limbal stem cell deficiency following topical mitomycin c treatement of conjunctival-corneal intraepithelial neoplasia[END_REF][START_REF] Kopp | Epiphora as a side effect of topical mitomycin C[END_REF] The purpose of this report is to describe our experience with topical MMC in the management of ocular surface neoplasia over the last 10 years. Data regarding treatment response, disease recurrence, and short and long-term complications are presented.

METHODS

A retrospective case note review was performed of all patients with a diagnosis of PAM, conjunctival melanoma, CCIN, conjunctival/corneal SCC and SGC, who had attended the ocular oncology service at a single centre between January 1998 and February 2009.

The majority of patients received the treatment regimen of one drop of topical MMC 0.04% QID for three weeks, with a three week period off, followed by another three weeks of MMC QID, regardless of whether treatment was primary, neoadjuvant or post-surgical adjuvant. For those receiving post-surgical adjuvant treatment, the regimen was commenced four weeks after surgery to allow wound healing. Topical steroids and lubricants were prescribed QID throughout the nine week period and beyond if required.

One patient had a similar regimen but with two week cycles, and two patients had a similar regimen but with four week cycles. All three of these variations occurred in 1998-1999 when MMC was first used in the department. A further four patients received intra-operative MMC only between 1998-2001, with application of 0.04% MMC for three minutes to the exposed base and margin tissue following surgical excision. Punctual occlusion was not used during MMC treatment. All treatments were supervised by a single ocular oncologist (EK).

The details of treatment indications are summarised in table 1.

Patients were examined at week four of treatment, and then at one -four weeks following completion of the course. Regular clinical review thereafter was arranged depending on the individual patient's needs. Outcome measures included initial clinical response to treatment, short-term complications, disease recurrence (including further tumour growth in lesions not showing complete regression following treatment), long-term complications (i.e. complications not resolving on treatment cessation) and long-term outcomes (i.e. clinical status at last review).

RESULTS

Fifty-eight patients (23 men and 35 women) who had received topical MMC chemotherapy were identified. The mean age at treatment was 63 years (range 24-91 years) and mean follow-up was 36 months (15 days to 10 years).

Histopathological diagnosis was available in 52 patients, which confirmed the clinical diagnosis in 51 patients. One patient with a clinical diagnosis of conjunctival melanoma and treated with intraoperative MMC, was found to have a conjunctival naevus on histological examination. The six patients with no histopathological diagnosis received MMC as a primary therapy for clinically diagnosed PAM.

Initial clinical response

Initial tumour response to treatment was graded as no-response, partial response (a decrease in the extent/pigmentation of the lesion), or complete clinical resolution (figure 1).

A favourable initial clinical response was evident in 93% (table 2 and figure 1). Of those that showed no initial clinical response, two were PAM. One required surgical excision twice, with neoadjuvant MMC and cryotherapy to the excision margins with the second excision.

The other patient defaulted follow-up then returned five years with increased lesion size which was excised and revealed a benign melanocytic proliferation. One melanoma showed no initial clinical response to primary treatment with MMC for recurrence, but due to serious co-morbidity, the patient died shortly after. Finally, one SCC showed no clinical response to excision with post-operative MMC, so required further excision with buccal mucous membrane graft to achieve disease control.

Short-term complications

Short-term complications of MMC therapy were recorded in 40 patients, and are summarised in table 3. The most common complication was allergy, 64% of which occurred during the second three week period of MMC drops. Overall, 67.5% of complications were reported during the second three week period of MMC treatment. Despite the frequency of short-term complications, treatment cessation due to intolerance was only merited in 4 patients (7%).

Disease recurrence A total of 14 patients developed recurrent pathology at a mean interval of 12.8 months following initial treatment with MMC (table 2).

Three patients with PAM developed recurrent disease. All were treated with further topical MMC. One now has stable disease while two are being observed for clinical response following recent completion of repeat treatment.

Five patients with conjunctival melanoma developed recurrent disease (three recurred as PAM and two as melanoma). All had received treatment as a surgical adjuvant (either to surgery performed at this centre or at the referring hospital), and four had initially shown compete clinical resolution. In addition, two had stopped treatment early due to allergy. At last review two were being observed with stable PAM, and three were clinically disease-free after additional interventions which included further topical MMC in one, and surgical excision with cryotherapy in two.

Two patients with SCC developed recurrent disease, both of whom had received postoperative adjuvant MMC, and had initially shown complete clinical disease resolution. One patient had stopped treatment early due to allergy. Both patients were clinically diseasefree at last review after additional interventions of excision with plaque brachytherapy in one, and excision then topical MMC then further excision with intra-operative MMC in the other.

Of four patients with SGC who developed tumour recurrence, three had received MMC as a primary treatment without surgical excision. One of these was because of misreporting of the biopsy by the referring hospital as CIN. Recurrence developed 24 months after treatment and was treated by surgical excision with skin graft, however excision margins were involved so the patient was awaiting further excision at the time of compiling this report. The second patient received MMC as a primary treatment following biopsy confirmation of the diagnosis, and showed only a partial clinical response. Further growth was evident at four months which was treated by surgical excision and despite clear excision margins, further recurrence occurred almost two years later. The third patient developed recurrent SGC 14 months following primary treatment with topical MMC. This was treated with an additional course of MMC. Further recurrence 41 months later required exenteration. Finally, the fourth patient had neoadjuvant MMC but developed recurrence due to incomplete surgical excision. Tumour control was achieved with further surgical excision. Apart from the second patient, the tumours in the other three had initially shown complete clinical resolution.

Long-term complications

Long-term complications following topical MMC therapy occurred in a total of 19 patients (33%). The most common complication was continuing mild keratoconjunctivitis which was managed satisfactorily with topical lubricants in all nine affected patients. Corneal epithelial changes due to presumed limbal stem cell deficiency (LSCD) occurred in seven patients, with three suffering persistent corneal erosions (delaying further treatment with topical MMC for recurrence in one patient). Two of the seven patients had MMC as a primary treatment so it is likely that MMC was the cause of this complication. The other five patients had surgical excision +/-cryotherapy in addition to MMC, making it impossible to attribute this complication solely to MMC. Epiphora due to punctal stenosis was documented in six patients, only one of whom had developed signs of allergy to the MMC drops. Finally, one patient developed lower lid ectropion following an allergic lid reaction to MMC. Several patients were also noted to have symblepharon and cicatricial conjunctival changes at the site of surgical excision which have not been included as long-term complications of topical MMC treatment as it was felt that they were surgical complications. Overall, visual acuity was maintained within two Snellen lines in 87%.

DISCUSSION

Use of topical MMC chemotherapy is now well established in the management of ocular surface neoplasia. For localised lesions, primary excision remains the treatment of choice, [START_REF] Shields | Surgical management of conjunctival tumors. The 1994 Lynn B. McMahon Lecture[END_REF] however neoadjuvant or post-surgical adjuvant treatment with MMC may further reduce the incidence of recurrence. [5,[START_REF] Chen | Mitomycin C as an adjunct in the treatment of localised ocular surface squamous neoplasia[END_REF][START_REF] Frucht-Pery | Topical mitomycin-C for partially excised conjunctival squamous cell carcinoma[END_REF] For larger lesions, neoadjuvant chemoreduction with MMC may increase the likelihood of subsequent surgical excision with clear margins, as demonstrated by one or our cases of SGC which showed clear excision margins following neoadjuvant treatment. This may also reduce amount of conjunctival resection required, thereby reducing the sequelae of extensive conjunctival resection. [START_REF] Shields | Chemoreduction with topical mitomycin C prior to resection of extensive squamous cell carcinoma of the conjunctiva[END_REF] Our results confirm the highly effective role of primary MMC treatment in the management of both PAM and CCIN. For the other pathologies with extensive involvement, primary MMC treatment may play a role in temporising the disease which may be particularly useful in patients with co-morbidities delaying surgery, and in those refusing radical surgery such as exenteration. This is illustrated by one of our cases of extensive SGC, where use of primary MMC treatment delayed the need for orbital exenteration by almost three and a half years.

Primary MMC treatment was also used to treat a clinically diagnosed conjunctival melanoma in one case in our series, which demonstrated a partial response. A subsequent biopsy revealed only PAM with atypia. As no pathological diagnosis was available pretreatment, it is possible that the lesion was never invasive. Poor results with primary MMC treatment of conjunctival melanoma has been reported previously, so our apparently good result should be interpreted with caution. [5] As seen by our results and those of others, primary MMC treatment is also unlikely to achieve disease control alone in SGC and SCC, particularly if the SCC is more than 4 mm thick. [START_REF] Shields | Chemoreduction with topical mitomycin C prior to resection of extensive squamous cell carcinoma of the conjunctiva[END_REF] Toxicity to topical MMC is widely reported, and remains a concern. In our series self-limiting complications such as hyperaemia, epiphora, keratoconjunctivits and allergy occurred frequently, however these were only severe enough to merit treatment cessation in 7% which is much lower than reported elsewhere (table 4). The routine prescription of potent topical steroids and lubricants throughout the duration of MMC treatment and beyond if necessary, which has not been described elsewhere, may explain this low treatment drop-out rate. Of the four that required treatment cessation, two had received a longer regimen of four weeks cycles. Three of the four patients stopped because of allergy and one because of uveitis and allergy. Uveitis as a complication of MMC has been reported previously, and occurred in a further two patients in our series. Other reported short-term complications such as corneal abrasion, corneal oedema, and pyogenic granuloma (requiring cryotherapy) occurred at low frequency.

Persisting complications following MMC treatment are of more concern, and occurred at a higher rate than we had anticipated. The most common problem was ongoing mild keratoconjunctivitis. Although previously reported, it is likely that this minor side-effect is underreported by both patient and doctor. Persisting corneal epithelial defects and corneal irregularities due to LSCD occurred in a significant number of patients. Although a similar incidence has been reported elsewhere (table 4), many other published studies have not reported LSCD to be a problem, including an earlier report from our unit. [START_REF] Kemp | Preoperative topical and intraoperative local mitomycin C adjuvant therapy in the management of ocular surface neoplasias[END_REF] However the mean follow-up for our seven patients with LSCD was 71 months, indicating that this is likely to be a problem that becomes more common with time. As is likely in other larger tertiary referral centre, patients are often discharged back to the referring hospital for observation after a period of satisfactory disease control, so it is possible that our data underestimates this serious complication.

Epiphora +/-punctual stenosis occurred in 10% of patients in our series, and as reported by others, was not predicted by the development of an allergic reaction to treatment. [20,[START_REF] Kopp | Epiphora as a side effect of topical mitomycin C[END_REF] This rate is similar to that reported in the largest published series of complications of MMC treatment by Khong and Muecke, where punctal occlusion was not employed. [20] As for KCS, it is likely that this too is underreported by patients, as indicated by one study which found that at one month post-treatment, 64% of patients had symptoms of epiphora. [START_REF] Kopp | Epiphora as a side effect of topical mitomycin C[END_REF] Routine punctal occlusion is not employed in our unit as it is felt that exposure of the lacrimal system to the chemotherapeutic effect of MMC is important in reducing the likelihood of tumour recurrence at this site. This may account for our incidence of punctal stenosis but there is little evidence that this is prevented by punctal occlusion. Furthermore, punctual occlusion increases the duration of exposure of the ocular surface to MMC which may be important in the development of intolerable side-effects which were low in our series. There is also evidence that the concentration of MMC in the aqueous humor increases linearly with MMC concentration and exposure time, with potentially increased risk of endothelial toxicity. [START_REF] Song | Mitomycin-C concentration in cornea and aqueous humor and apoptosis in the stroma after mitomycin-C application[END_REF] As our recurrence rates are comparable to other reported series (table 4) it is unlikely that any prolonged contact with MMC achieved with punctual occlusion is beneficial to tumour control.

In summary, the results of our series demonstrate the effective role of topical MMC in the management of a variety of ocular surface neoplasia, reducing the need for radical surgical intervention. As in other studies, short-term complications were fairly common but largely self-limiting. However, in accordance with studies with longer follow-up, limbal stem cell deficiency appears to be a significant long-term risk of MMC treatment. This serious complication should therefore be taken into consideration when contemplating use of topical MMC. In view of the malignant potential of some of these tumours, this may be an acceptable risk when compared to the consequences of previous treatment modalities. Recurrence rates were calculated from those that showed an initial response to treatment.

Overall recurrence rate was calculated from those that showed an initial response, excluding the conjunctival naevus (n=53).

Figure 1 .

 1 Figure 1. (a) Pagetoid involvement of the cornea from an upper lid SGC. (b) The same patient showing complete clinical resolution of the corneal involvement after one cycle of topical MMC, leaving only deep stromal vascularisation. (c) PAM of the nasal conjunctiva. (d) The same patient showing only minimal residual pigmentation at the same site following one course of topical MMC.

Table 1 .

 1 Summary of the indications for treatment by diagnosis.

	Indications		PAM	Melanoma	CIN SCC	SGC	total
	primary treatment		15	1	2		3	21
	Neoadjuvant		1	3	3	2	2	11
	adjuvant intra-op			3			1	4
	post-op adjuvant			11	1	2		14
	for recurrence -neoadjuvant		1	1		1	3
	primary treatment for		1	3	1			5
	recurrence
	total		17	22	8	4	7	58
	clinical response	PAM	Melanoma	CIN	SCC	SGC	naevus	total
	no response	2	1		1				4
	partial	13	4	1		1			19
	complete	2	16	7	3	6	1		35
	mean follow-up	47	34	20	50	28.5	1		36
	(range) months	(4-120)	(2-112)	(1-49)	(4-82)	(0.5-54)		(0.5-120)
	overall	3/15	5/20	0/7	2/3	4/7	0/1	14/53
	recurrence	(20%)	(25%)		(67%)	(57%)			(26%)
	mean time to	16	14		3 . 5	13.5			12.8
	recurrence	(14-18)	(1-30)		(2-5)	(4-24)			(1-30)
	(range) months
	Recurrence for eyes	3/15	4/16	0/4	2/2	4/5			13/42
	with minimum 12	(23%)	(25%)		(100%)	(80%)			(31%)
	months follow-up

Table 2 .

 2 Summary of initial clinical response and recurrence by diagnosis. For tumours treated with adjuvant MMC, complete or partial clinical response refers to overall response to both surgery and MMC. Mean times to recurrence, including the range, are included.

Table 3 .

 3 Short-term complication by symptom onset during first or second 3 week period of topical MMC treatment.

Table 4 .

 4 Comparison of our study group outcomes compared to other published outcomes.

MM = conjunctival melanoma.


Louise E Allen
email: louise.allen@addenbrookes.nhs.uk

Edel M Cosgrave

James P Kersey

Uma Ramaswami

Consultant Miss Louise Allen

Fabry disease in children: correlation between ocular manifestations, genotype and systemic clinical severity

Background/aims: Fabry disease is an X-linked lysosomal disorder associated with severe multi-organ failure and premature death. This study aims to determine the prevalence of ophthalmic manifestations in children with the condition and investigate the correlation with genotype and systemic disease severity.

Methods:The records of 26 children from 18 pedigrees with Fabry disease undergoing regular ophthalmic and systemic examination were reviewed. All pedigrees underwent GLA gene sequencing to determine genotype. Correlations between ocular and systemic phenotype and genotype were investigated.

Results: Corneal verticillata occurred in 50% of the children in this study (95% confidence interval 29-79%). Children with ophthalmic manifestations were more likely to have loss-of-function GLA mutations (p=0.003). Retinal vascular tortuosity was seen in seven children (27%) all of whom had systemic symptoms suggestive of autonomic neuropathy, such as diarrhoea and syncope. These symptoms seemed less prevalent in children without retinal vascular changes, although this did not reach statistical significance (p=0.134).

Conclusion

: Ophthalmic manifestations of Fabry disease are common, even in young children, with loss-of-function GLA gene mutations. Although the limited sample size possibly prevented statistical significance, systemic symptoms of autonomic neuropathy often coexist with retinal vascular changes and may share the same pathogenesis.

INTRODUCTION

Fabry disease (OMIM 301500) is an X-linked inherited lysosomal storage disorder associated with severe multi-organ dysfunction and premature death. [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF] Fabry disease is hemizygous in males and heterozygous in females, but both genders can be severely affected, although the disease is usually slower to progress and is more variable in females. [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF][START_REF] Whybra | Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes[END_REF][START_REF] Whybra | A 4-year study of the efficacy and tolerability of enzyme replacement therapy with agalsidase alfa in 36 women with Fabry disease[END_REF][START_REF] Galanos | Clinical features of Fabry disease in Australian patients[END_REF][START_REF] Kampmann | Cardiac manifestations of Anderson-Fabry disease in heterozygous females[END_REF] The condition is rare, with an estimated incidence of up to1 in 40,000 male live births. [START_REF] Meikle | Prevalence of lysosomal storage disorders[END_REF] GLA gene mutations reduce the activity of α-galactosidase A, resulting in globotriaosylceramide (Gb 3 ) deposition in a variety of cells, including those of the kidney, autonomic and cardiovascular system, as well as the cornea. [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF][START_REF] Branton | Natural history of Fabry renal disease: influence of α-galactosidase A activity and genetic mutations on clinical course[END_REF] Ophthalmologists are a key group in the identification and referral of patients with Fabry disease. Eye examinations are relatively simple and represent a noninvasive method for assisting in the clinical diagnosis of patients with potential Fabry disease. [START_REF] Sodi | Ophthalmological manifestations of Fabry disease[END_REF][START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF] Ophthalmological manifestations of Fabry disease result from the progressive deposition of glycosphingolipids in various ocular structures, although these do not usually cause ocular symptoms or visual impairment. [START_REF] Sodi | Ophthalmological manifestations of Fabry disease[END_REF] The most frequently reported eye abnormalities are corneal verticillata (corneal opacities), conjunctival and retinal vascular abnormalities (vessel dilatation and tortuosity) and lens opacities (Figures 1 and2). [START_REF] Sodi | Ophthalmological manifestations of Fabry disease[END_REF][START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF] . Ophthalmological signs are usually present in children before serious systemic symptoms develop. [START_REF] Ramaswami | Clinical manifestations of Fabry disease in children: data from the Fabry Outcome Survey[END_REF] The relative rarity and variability of the condition make diagnosis difficult and many patients suffer from delayed diagnosis or misdiagnosis for years. In the Fabry Outcome Survey (FOS), of 366 patients in all age groups, the mean time between onset of symptoms and diagnosis was 13 years for men and 16 years for women. [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF] In children (< 18 years), there was an approximate 3-year delay between symptom onset and diagnosis. [START_REF] Ramaswami | Clinical manifestations of Fabry disease in children: data from the Fabry Outcome Survey[END_REF] Accurate and rapid diagnosis of Fabry disease, particularly in children, is urgently needed now that effective treatment with enzyme replacement therapy (ERT) is available. Replacement α-galactosidase A (agalsidase alfa: Replagal, Shire Human Genetic Therapies, agalsidase B: Fabrazyme , Genzyme Europe) therapy has been demonstrated to clear tissue globotriaosylceramide and to improve clinical endpoints, including stabilizing renal function, reducing pain, improving cardiac structure and function, improving gastrointestinal symptoms and stabilizing mild-to-moderate hearing loss. [START_REF] Beck | Fabry disease: overall effects of agalsidase alfa treatment[END_REF][START_REF] Schaefer | Enzyme replacement therapy for Fabry disease: A systematic review of available evidence[END_REF][START_REF] Hoffmann | Gastro-intestinal symptoms in 342 patients with Fabry disease: prevalence and response to enzyme replacement therapy[END_REF][START_REF] Hughes | Effects of enzyme replacement therapy on the cardiomyopathy of Anderson-Fabry disease: a randomised, double-blind, placebo controlled trial of agalsidase alfa[END_REF][START_REF] West | Agalsidase alfa and kidney dysfunction in Fabry disease[END_REF] It may now therefore be possible to halt and even reverse the progress of Fabry disease before irreversible organ damage occurs.

Symptoms of Fabry disease, particularly acroparaesthesiae (numbness, pain or tingling in the extremities) and altered temperature sensitivity, altered bowel habits and abdominal pain, together with angiokeratoma, hypohidrosis, lethargy, and ophthalmological and auditory signs usually present in childhood (< 10 years), with life-threatening renal, cardiac or neurological complications in adulthood. [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF][START_REF] Meikle | Prevalence of lysosomal storage disorders[END_REF][START_REF] Ramaswami | Clinical manifestations of Fabry disease in children: data from the Fabry Outcome Survey[END_REF][START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF] Resultant renal failure, cardiomyopathy and cerebrovascular disease lead to premature death, on average 15 years earlier in women and 20 years earlier in men compared with the general population. [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF] To extend these studies in children, we investigated the relationship between genotype, ophthalmic phenotype and the severity of the systemic manifestations in children with the diagnosis of Fabry disease attending our centre. The aim was to assess whether ocular involvement is associated with a more severe systemic phenotype in affected children, and whether this is related to genotype. ERT is considered by the paediatric metabolic team if a child has uncontrolled pain leading to a need to alter lifestyle, or pain that interferes with quality of life and has one or more of the following manifestations: severe asthenia, lethargy, gastroenterological symptoms such as pain, vomiting or altered bowel habit, abnormal MRI, episodic vertigo, intra-ventricular conduction defects, and/or poor growth that cannot be accounted for by another cause. [START_REF] Da Hughes | Guidelines for the diagnosis and management of Anderson-Fabry Disease[END_REF] Systemic phenotyping was determined by multi-system history, examination and laboratory results. Ocular and systemic phenotypes were correlated with genotype, gender, age, systemic manifestations of the disease and commencement of ERT.

MATERIALS AND METHODS

The

RESULTS

We reviewed data from 26 children from 18 pedigrees (12 hemizygous boys of median age 5 years and 14 heterozygous girls of median age 11 years (range 1-years) were already receiving ERT at the time of the first ophthalmic examination.

Molecular Genetic Results

Thirteen different GLA gene mutations were identified (Table 2). Loss-of-function mutations affected 17 children; mutations predicted to give residual enzymatic activity affected nine children.

Systemic Examination

The most frequent systemic manifestations of Fabry disease were gastrointestinal (n = 20 patients) neurological (n = 14) and cardiac (n = 15) and auditory (n = 16) abnormalities. All other abnormalities were reported for less than 5 patients.

Ophthalmic manifestations were found in 76% of patients predicted to have no enzyme function and in only 1 patient with a genotype predicted to give some residual enzyme function (Table 3) (Fisher's exact test p=0.03). However, patients predicted to have some residual enzyme function had a similar prevalence of neurological, gastrointestinal and auditory symptoms as those with loss-of-function mutations. Cardiac and renal symptoms were actually more common in the group with predicted residual enzyme function.

DISCUSSION

Fabry disease is a rare condition and, in this context, the 26 children and adolescents included in the current study are a significant cohort of young patients. The data from the current study agree with and add to the literature on reported ophthalmological manifestations of Fabry disease.

The FOS study demonstrated that the most common ocular manifestation of Fabry disease is corneal verticillata (occurring in approximately 70% of adult patients) and that its presence does not correlate with Fabry illness severity measured by disease progression, renal function and cardiac size. [START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF] In our study, corneal verticillata were seen in 50% of children studied (95% CI 29-79%), the youngest being a 2 year old heterozygous female. The children with verticillata did not tend to be older than the group without corneal changes.

The FOS study showed a prevalence of retinal vascular tortuosity of 49% in males and 22% of females and of Fabry cataract in 23% males and 10% females; these features are thought to become more common with age. [START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF][START_REF] Ramaswami | Clinical manifestations of Fabry disease in children: data from the Fabry Outcome Survey[END_REF] Vessel tortuosity on its own is not diagnostic for Fabry disease but has been recognised as being predictive of more severe illness severity. [START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF][START_REF] Fumex-Boizard | Relation between ocular manifestations and organ involvement in ten patients with Fabry disease[END_REF] Retinal vascular tortuosity was present in 27% of the children in this study and was identified in a child of only 4 years of age. Whereas all the children who had evidence of retinal vascular tortuosity also had gastro-intestinal and neurological symptoms, a minority of those without retinal vascular changes manifested these symptoms of autonomic neuropathy. This did not meet statistical significance (Fisher's exact test p=0.134) in our study, possibly due to the small sample size, but does hint of a possible association between these features.

The pathophysiology of retinal vascular dilatation and tortuosity in Fabry disease is not fully understood. Autonomic neuropathy has been implicated as the cause of the diarrhoea, hypohydrosis and syncope seen in Fabry patients. [START_REF] Laaksonen | Neuropathic symptoms and findings in women with Fabry disease[END_REF] Autonomic neuropathy secondary to diabetes has been reported to cause both bulbar and retinal vascular dilatation and tortuosity, as has cervical sympathectomy, due to the detrimental effect on retinal vascular auto-regulation. [START_REF] Lanigan | The effect of cervical sympathectomy on retinal vessel responses to systemic autonomic stimulation[END_REF][START_REF] Lanigan | Responses of retinal circulation to systemic autonomic stimulation in diabetes mellitus[END_REF][START_REF] Tesfaye | Arterio-venous shunting and proliferating new vessels in acute painful neuropathy of rapid glycaemic control[END_REF][START_REF] Owen | Diabetes and the tortuosity of vessels of the bulbar conjunctiva[END_REF] Retinal and bulbar conjunctival vessel dilatation and tortuosity are therefore likely to be a marker for autonomic neuropathy in Fabry patients.

Loss-of-function mutations affected approximately two-thirds of patients in the present study, while mutations predicted to give residual enzymatic activity affected about one-third of patients. Although ophthalmic findings were much more commonly associated with loss-of-function mutations (Fishers exact test p=0.03), the prevalence of cardiac and renal systemic manifestations was higher in genotypes with predicted residual enzymatic activity.

Although the absence of ophthalmic signs does not appear to correlate with milder systemic disease, those children with absent eye signs but known systemic manifestations of Fabry disease may be predicted to have a missense mutation causing residual enzyme activity rather than a loss-of-function mutation.

Ophthalmological examination including slit-lamp examination should be conducted in young patients with symptoms of Fabry disease (e.g. neuropathic pain, diarrhoea, tinnitus, vertigo). Once other causes of corneal verticillata (such as long term therapy with choroquine or amiodarone) have been excluded, the presence of cornea verticillata is diagnostic of Fabry disease. The additional presence of retinal vascular tortuosity indicates that autonomic neuropathy is likely and these patients may already have potentially severe systemic manifestations.

Serial ophthalmological examinations may also be useful in monitoring the response to ERT. Quantification of severity of corneal verticillata is currently highly subjective. [START_REF] Sodi | Ocular manifestations of Fabry's disease: data from the Fabry Outcome Survey[END_REF] The presence of retinal vascular dilatation and tortuosity was assessed subjectively in this study and generally this has been shown to be a reliable form of assessment. [START_REF] Owen | Assisted Image Analysis of the Retina (CAIAR) program[END_REF] To date there have been no large population studies determining the prevalence of retinal vascular dilatation and tortuosity in the normal population although it is felt that these changes, even in childhood, may be a potential predictor for vascular disease in later life. [START_REF] Owen | Assisted Image Analysis of the Retina (CAIAR) program[END_REF]

  Paediatric Metabolic Unit at Cambridge University Hospitals NHS Foundation Trust (Addenbrooke's Hospital) holds a database of children with Fabry disease seen in the Department. All pedigrees are genotyped by GLA gene sequencing and the children have annual ophthalmic and biannual systemic examinations. We analysed the first recorded ophthalmic examination and clinical findings for eight different systems for children with Fabry disease undergoing their biannual review. The ophthalmic examination included: visual acuity assessment, refraction and slit-lamp examination for conjunctival and retinal vascular tortuosity, corneal haze/verticillata and lens opacities. Anterior segment and retinal photography were performed. Retinal vascular tortuosity was assessed by a single experienced ophthalmologist (LEA) studying the calibre and directness of path of vessels using digital retinal photographs. Systemic examination included a general assessment performed by a single experienced metabolic paediatrician (UR) with specific questioning regarding symptoms, and full physical examination. Radiological evaluation of cardiac, renal and neurological function, and audiometry were performed annually at the same centre by experienced personnel.

26 CONCLUSIONFigure 1 :Figure 2 :

 2612 Figure 1: slit-lamp photograph of corneal verticillata

Table 1 .

 1 GLA genotypes detected in patients with Fabry disease The median age range of the children with verticillata was 8.1 years (range 2-15 years) whilst that of the group without verticillata was 10.9 years range (1-15 years).

	Mutation type Predicted effect	N = 26	Genotype (n)
	Nonsense	Loss-of-function	5	p.W209X (2)
				p.R301X (2)
				p.R227X (1)
	Deletion	Loss-of-function	1	c.1088delG (1)
	Missense	Predicted loss-of-	11	p.R49S (4)
		function
				p.N298H (1)
				p.P205T (1)
				p.C520T (2)
				c.902G>A (1)
				c.319C>T (1)
				p.T950C (1)
	Missense	Predicted residual	9	p.N215S (7)
		activity
				p.A143T (2)

Ophthalmic Examination

Thirteen of the 26 children (50%) were found to have corneal verticillata, the youngest being a girl 2 years of age (95% confidence interval 29-79%). None had severe corneal manifestations and, of the 7 children with moderate changes, 6 were heterozygous females.

Table 2 .

 2 Systemic features in children with retinal vascular tortuosity. (* denotes

	patients on ERT)
	Age(yrs) Genotype NeurologicalGastrointestinal Cardiac Renal Auditory
	13.5*	1088delG	+	+	-	+	+
	12.8	R301X	+	+	-	-	-
	8.8*	R49S	+	+	+	-	+
	4.0	P205T	+	+	+	-	+
	15.0*	C520T	+	+	+	-	+
	13.5*	C520T	+	+	+	-	+
	14.7	R301Q	+	+	+	-	-

Table 3 .

 3 Comparison of frequency of systemic features with genotype groups.

	Predicted	N=26 Eye	NeurologicalGastrointestinal Cardiac Renal Auditory
	enzyme		signs
	activity
	None	17 13	10	13	8	2	10
			(76%)	(59%)	(76%)	(47%)	(12%)	(59%)
	Residual 9	1	5	7	7	2	6
			(11%)	(55%)	(78%)	(78%)	(22%)	(67%)

ACKNOWLEDGEMENTS

We thank Clare Taylor and Alison Hough, paediatric metabolic specialist nurses for their help with tabulating the clinical data and Serena Nik-Zainal for her help with interpretation of the molecular genetic results.


Mahiul Mk Muqit Mrcophth

PhD George R Marcellino

Jane Cb Gray

Rita Mclauchlan

PhD David B Henson

Lorna B Young

Niall Patton

Stephen J Charles Frcophth

George S Turner Frcophth

MD Paulo E Stanga

Pain Responses of Pascal ® 20 ms Multi-spot and 100 ms Single-spot Panretinal Photocoagulation: MAnchester PAScal® Study, MAPASS report 2

Keywords: Pascal ® panretinal photocoagulation, pain responses, conventional laser, proliferative diabetic retinopathy, nociception

Aims:

To evaluate pain responses following Pascal ® 20ms multi-spot and 100ms single-spot panretinal photocoagulation (PRP).

Methods:

Single-center randomized clinical trial. 40 eyes of 24 patients with treatment-naive proliferative diabetic retinopathy randomized to 20ms-PRP and 100ms-PRP under topical 0.4% oxybuprocaine. A masked grader used a pain questionnaire within one-hour (Numerical pain score: NPS) and one-month after treatment (Numerical headache score: NHS). Primary outcome measure was NPS immediately post-PRP. Secondary outcome measures were mean NHS scores and levels of photophobia reported within 4-weeks of primary PRP.

Results:

Mean laser fluence was significantly lower using 20ms-PRP (4.8 J/cm 2 ) compared to 100ms-PRP (11.8 J/cm 2 ; p<0.001). Mean NPS scores for treatment were 2.4±2.3 (mild) for 20ms-PRP group compared to 4.9±3.3 (moderate) in 100ms-PRP group, a significant difference (95%CI -4.3 to -0.68;p=0.006). Mean NHS score within 1-month was 1.5±2.7 in 20ms-PRP group compared to 3.2±3.5 in 100ms-PRP group (p<0.05). The median duration of photophobia after 20ms-PRP was 3-hours, and significantly less compared to 100ms-PRP after which 72-hours of photophobia was reported (p<0.001).

Conclusions:

Multi-spot 20ms-PRP was associated with significantly lower levels of anxiety, headache, pain, and photophobia compared to 100ms single-spot PRP treatment. Possible

reasons include lower fluence, shorter-pulse duration, and spatial summation of laser nociception with multi-spot Pascal® technique.

Laser panretinal photocoagulation (PRP) standards for treating proliferative diabetic retinopathy (PDR) were established by the Diabetic Retinopathy Study 20 years ago. [START_REF] Retinopathy | Photocoagulation treatment of proliferative diabetic retinopathy. The second report of diabetic retinopathy study findings[END_REF] The conventional long pulse durations (100-200 milliseconds,ms) used for PRP may be associated with significant pain and laser-intolerance by patients. [START_REF]Local Anaesthesia for Intraocular Surgery[END_REF] A number of pre-laser injection strategies have been investigated. Subconjunctival and sub-Tenon injections may be effective, but sharp needle peri/retrobulbar anesthesia is rarely used due to potential sight-threatening complications. [START_REF]Local Anaesthesia for Intraocular Surgery[END_REF][START_REF] Stevens | No-needle one-quadrant sub-tenon anaesthesia for panretinal photocoagulation[END_REF][START_REF] Wu | Interventions for relieving pain associated with panretinal photocoagulation: a prospective randomized trial[END_REF][START_REF] Hay | Needle penetration of the globe during retrobulbar and peribulbar injections[END_REF] Oral acetaminophen, diazepam, diclofenac, mefenamic acid, and paracetamol have all been studied. [START_REF] Wu | Interventions for relieving pain associated with panretinal photocoagulation: a prospective randomized trial[END_REF][START_REF] Zakrzewski | Oral versus topical diclofenac for pain prevention during panretinal photocoagulation[END_REF][START_REF] Vaideanu | Double masked randomised controlled trial to assess the effectiveness of paracetamol in reducing pain in panretinal photocoagulation[END_REF] Entonox and intramuscular ketorolac tromethamine have also been studied pre-laser. [START_REF] Wu | Interventions for relieving pain associated with panretinal photocoagulation: a prospective randomized trial[END_REF][START_REF] Cook | Entonox as an analgesic agent during panretinal photocoagulation[END_REF] To date, oral diclofenac has been shown to be effective in reduction of post-laser pain response. [START_REF] Zakrzewski | Oral versus topical diclofenac for pain prevention during panretinal photocoagulation[END_REF] Randomized trials of pretreatment analgesia for PRP using pain scales report mean placebo group pain scores that range from 37.3 to 53.1. [START_REF] Wu | Interventions for relieving pain associated with panretinal photocoagulation: a prospective randomized trial[END_REF][START_REF] Zakrzewski | Oral versus topical diclofenac for pain prevention during panretinal photocoagulation[END_REF][START_REF] Weinberger | Analgesic effect of topical sodium diclofenac 0.1% drops during retinal laser photocoagulation[END_REF][START_REF] Esgin | Topical ketorolac 0.5% for ocular pain relief during scatter laser photocoagulation with 532 nm green laser[END_REF] Pain scores of 31-69 fall within the moderate range of pain. [START_REF] Kelly | Does the clinically significant difference in visual analog scale pain scores vary with gender, age, or cause of pain?[END_REF] Recently, clinicians have undertaken conventional laser PRP parameters with reduced 50ms pulse duration. [START_REF] Brucker | Observational study of the development of diabetic macular edema following panretinal (scatter) photocoagulation given in 1 or 4 sittings[END_REF] Reductions in pain response have been reported with different pulse waveforms and 20ms pulse duration PRP. [START_REF] Friberg | Alteration of pulse configuration affects the pain response during diode laser photocoagulation[END_REF][START_REF] Al-Hussainy | Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times[END_REF] The Pascal ® (Pattern Scanning Laser) Photocoagulator uses a brief pulse duration combined with rapid raster scan that allows effective multi-spot applications. [START_REF] Blumenkranz | New Instrument: Semiautomated Patterned Scanning Laser for Retinal Photocoagulation[END_REF][START_REF] Sanghvi | Initial Experience with the Pascal ® Photocoagulator: a Pilot Study of 75 Procedures[END_REF] Furthermore, this technique may be less damaging to inner retina with reduced collateral thermal diffusion. [START_REF] Muqit | Fundus Autofluorescence and Fourier-Domain Optical Coherence Tomography Imaging of 10 and 20 millisecond Pascal ® Retinal Photocoagulation Treatment[END_REF] The conception of this study was based on the following observations: pain may reduce laser uptake during photocoagulation, pain-related anxiety post-PRP may reduce compliance and further delay application of laser in sight-threatening diabetic retinopathy, and reduced pulse laser may reduce pain.

The primary aim of this study was to compare the pain responses under topical anesthesia of multi-spot 20ms-PRP and single-spot 100ms-PRP burns in PDR. Our secondary aims included an evaluation of the characteristics of pain responses, subjective responses post-laser, and evaluation of patient's experiences related to laser-induced inflammation.

Materials and Methods

A prospective, randomized clinical trial was carried out with patients treated at Manchester Royal Eye Hospital (MREH) over a 1-year period. The study protocol received Research Ethics Committee approval, and informed written consent was obtained from all participants. Data and safety monitoring was provided by an independent panel at both the University of Manchester and the Research Office at MREH. The inclusion and exclusion criteria are outlined in Table 1.

Literature searches were carried out in the PubMed and MEDLINE databases between January 1993 and June 2009 to uncover all previously published articles describing pain modification techniques for PRP using the search terms panretinal photocoagulation, laser photocoagulation, pain, and laser pulse duration.

The randomization sequence was generated using randomly permuted blocks, and a randomization table was created for the 40 subjects. After computerized randomization procedure, sequentially-numbered, opaque, sealed envelopes were generated by the masked trial statistician. The treatment allocation envelopes remained concealed until interventions were assigned. Once a participant consented to the study, a research trial coordinator who was blinded to the treatment opened the sealed envelope and assigned participants to each group. The treatment was not blinded to the treating investigator or study participant. Patients underwent either unilateral or bilateral PRP according to randomization treatment allocation. In cases of bilateral study eye eligibility, the right eye was randomized first with treatment allocation, followed by independent randomization and allocation of the left eye. Eyes were randomized into one of two groups, 20ms-PRP or 100ms-PRP.

The primary efficacy endpoint was the NPS score for each group immediately post-PRP. Main secondary efficacy endpoints included duration of pain responses during PRP, NHS score at 1-month, affective changes post-treatment, and levels of photophobia post-PRP.

Safety endpoints included all adverse events reported spontaneously by study participants, elicited by investigators, or observed by investigators. Adverse events were graded as mild, moderate, or severe and were assessed as being either related to the laser intervention or unrelated to the laser treatment. We recorded all serious adverse events whether deemed related to the treatment or not, as per ethical and good clinical practice.

Pre-laser anaesthesia used up to 5 drops of topical 0.4% oxybuprocaine hydrochloride applied over 5 minutes. Photocoagulation was applied using techniques outlined in Table 2 with a Mainster 165 PRP lens. Before trial commencement, two experienced retinal specialists at MREH externally validated the laser technique of the treating investigator (MM), and laser training was certified according to the study laser protocol and good clinical practice guidelines. Threshold laser burn intensity was standardized for all eyes, and this was checked at 1-hour post laser using fundus photographs.

This pain assessment was part of MREH guidelines for nurses and was adapted from McGill Pain Questionnaire. [START_REF] Melzack | Textbook of Pain[END_REF] This measurement tool has been externally validated previously, and is routinely used at MREH as a reliable and sensitive measure of pain responses. [START_REF] Sanghvi | Initial Experience with the Pascal ® Photocoagulator: a Pilot Study of 75 Procedures[END_REF][START_REF] Mc Caffery | Pain: Clinical Manual[END_REF] An examiner, masked to the treatment, used a standard questionnaire to assess the pain responses, characteristics of responses, and levels of affect.

Participants were asked to rate the level of pain related to the treatment session using the numerical pain scale (NPS). The NPS allows the patient to rate the pain intensity on a numbered scale, from 0 to 10. Zero is absence of pain, 1-3 is mild pain, 4-6 is moderate pain, and 7-10 is designated severe pain. In subjects undergoing multiplesession PRP, we recorded the mean NPS score over 3 treatment sessions. The duration of pain responses was reported using a 3-point scale: first half of treatment, second half of treatment, and full duration of treatment.

The laser illumination lamp intensity was adjusted to minimise unnecessary photophobia or discomfort. For patients receiving bilateral PRP, the first eye was treated followed by pain assessment, and after a delay, the second eye was treated followed by pain assessment. This method ensured that treatment of second eyes did not elicit an exaggerated or diminished pain response depending on the nature and fluence of the first eye's treatment.

At one-month post-PRP the same masked examiner carried out a second questionnaire-based interview to assess headache responses, and used a numerical headache scale (NHS), analogous to the NPS. Patients were asked about presence of photophobia, photopsia, and affective responses (anxiety, mood disturbances) within the preceding 4-weeks. The responses were recorded as increased, reduced, or unchanged compared to the day of primary PRP laser. We scored these responses using the ratio of increased versus decreased responses as the (I:R) ratio.

We performed statistical analyses using STATISTICA version 6 (StatSoft, Inc [2003], www.statsoft.com). We used the 2-tailed t test to explore mean NPS and NHS scores, and duration of photophobia following treatment at specified time-points. The null hypothesis was rejected for P values less than 0.05.

The sample size was based on the following assumptions: the test of significance should be 2-sided, with a significance level of 5%. Using a standard deviation of 2.0, if the true difference in the mean NPS response of matched pairs is 1.5, we will need to study 20 pairs of subjects (20 experimental and 20 control subjects) to be able to reject the null hypothesis that this response difference is zero with probability (power) 0.8.

The Type I error probability associated with this test of this null hypothesis is 0.05. A 1.5-point mean NPS difference was chosen since this represented a realistic value within the ranges of pain scores published in the literature for laser PRP studies to achieve significance. [START_REF] Zakrzewski | Oral versus topical diclofenac for pain prevention during panretinal photocoagulation[END_REF][START_REF] Vaideanu | Double masked randomised controlled trial to assess the effectiveness of paracetamol in reducing pain in panretinal photocoagulation[END_REF][START_REF] Weinberger | Analgesic effect of topical sodium diclofenac 0.1% drops during retinal laser photocoagulation[END_REF][START_REF] Esgin | Topical ketorolac 0.5% for ocular pain relief during scatter laser photocoagulation with 532 nm green laser[END_REF]

Results

A total of 40 eyes of 24 patients (20 in each arm) were recruited into the study between June 23 rd 2008 and July 10 th 2009 (Figure 1). Complete data capture from study questionnaires were obtained for 40 eyes all visits. The age profiles were similar in both groups with mean 46 years. No patient required additional PRP treatment within 1-month of the primary PRP. Patient demographics and laser parameters are presented in Table 3.

Examples of threshold laser burns intensities at 1-hour are shown in Figure 2. Laser fluence was significantly greater in 100ms-PRP compared to 20ms-PRP (average values 11.8 and 4.8 J/cm 2 ; p=0.0001), however the intensity of photocoagulation burns was equivalent in both groups.

The mean NPS score immediately post-laser was 2.4 ± 2.3 for 20ms-PRP group, compared to 4.9 ± 3.3 (100ms-PRP) categorized as moderate severity of pain (Figure 3). [START_REF] Kelly | Does the clinically significant difference in visual analog scale pain scores vary with gender, age, or cause of pain?[END_REF] The pain following multi-spot 20ms-PRP was mild, and this was significantly lower than the single-spot 100ms-PRP (95% CI -4.3 to -0.68; p=0.006). The mean NHS score within one month of treatment was 1.5 ± 2.7 in the 20ms-PRP group compared to 3.2 ± 3.5 (100ms-PRP group, Figure 4). The difference in NHS scores between groups was significant (95% CI -3.7 to 0.3; p=0.045).

During treatment, the mean contact lens-related NPS score was 3 in 4 eyes (20%) for 20ms-PRP group, and mean NPS was 6 in 4 eyes (20%) for the 100ms-PRP group.

Pain related to the laser contact lens was reported in 20% total study eyes, and this source of pain was reported as either a "gritty" or "foreign-body" sensation in all cases. The characteristics of side-effects related to pain responses are presented in table 4. As shown in table 2, subjects in 100ms-PRP group reported significantly increased levels of anxiety compared to Pascal® 20ms-PRP treatment (p<0.05). At 1-month, patients in both arms reported equivalent anxiety and mood changes, related to apprehension regarding the future beneficial outcomes of laser treatment rather than any pain responses.

Pain responses reported by group 20ms-PRP occurred during the first half of treatment in 11 (55%) and during the full duration in 3 (15%). In the 100ms-PRP group, pain responses were reported to last the full duration of treatment in 16 eyes (80%). The remainder did not report any pain during treatment (30% 20ms-PRP, 20% 100ms-PRP).

The data for levels of photophobia reported within 1-month of PRP in both groups was skewed, and median values were analyzed for comparison to achieve a better central tendency than arithmetic mean scores (Table 5). Median duration of photophobia after 20ms-PRP was 3 hours, and this was significantly less compared to 100ms-PRP (72 hours, p<0.001). The effects of PRP on driving and work performance, reading ability, watching television, and navigation during night-time did not show any significant differences or results between either group (Table 5). There were no significant differences in physiological responses designated by I:R ratios for either treatment group. There were no ocular complications during the immediate or short-term, and no reported adverse or serious adverse events. In the 20ms-PRP group, there were no signs of intraretinal haemorrhage or blood vessel damage from photocoagulation burns.

Discussion

Our results have demonstrated that single-session, multi-spot 20ms-PRP using topical 0.4% oxybuprocaine is significantly more comfortable for patients with PDR compared to conventional multiple-session 100ms-PRP.

Although the retina is devoid of pain sensitivity, ocular pain and photophobia are frequently reported post-laser. Laser-induced eye nociception may be related to thermal effects within choroid, stimulation of ciliary nerves within suprachoroidal spaces, thermal diffusion to nerve fiber layer, or perhaps direct photocoagulation of the long posterior ciliary nerve. [START_REF] Belmonte | Neurbiology of ocular pain[END_REF] Laser photocoagulation may produce a thermal rise in outer retina. Leukocyteendothelial cell interactions can lead to inflammatory maculopathy post-PRP in animal models, with increased cytokine release and retinal capillary hyper-permeability. [START_REF] Nonaka | Inflammatory response after scatter laser photocoagulation in nonphotocoagulated retina[END_REF] Recent work with rabbit retina demonstrated that 100ms pulse duration burns produced full-thickness retinal injury on pathological sections. [START_REF] Paulus | Healing of retinal photocoagulation lesions[END_REF] Laser photocoagulation may stimulate ocular nociception through inflammatory, mechanical, and thermal stimulation. A significantly higher fluence was required for 100ms-PRP that will produce higher levels of intraretinal inflammation. Longer standard pulse duration was associated with higher pain scores and greater photophobia post-laser.

Local ocular inflammatory responses may enhance outer retinal neurogenic inflammation in ocular nociceptive terminals and higher order neurons. [START_REF] Belmonte | Neurbiology of ocular pain[END_REF] The conventional 100ms-PRP produced pain for the full duration of PRP, whereas the multi-spot Pascal® 20ms pattern arrays triggered pain responses during the first half of PRP. This temporal aspect of pain may be related to spatial summation of pain associated with shorter-pulse Pascal®. A 20ms pulse reduces the time required for intraretinal tissue thermal changes to occur. Better localisation of 20ms burns within outer retina will minimise heat diffusion towards the retinal nerve layer and choroid. [START_REF] Muqit | Fundus Autofluorescence and Fourier-Domain Optical Coherence Tomography Imaging of 10 and 20 millisecond Pascal ® Retinal Photocoagulation Treatment[END_REF] Furthermore, the 1.5 burns spot-spacing means each 20ms burn within the array is applied to the retina in a scatter technique. The Pascal® rapid raster application of multiple spots may lead to habituation of pain associated with the laser arrays during PRP. The interactions between these spatial summation and temporal aspects of repetitive noxious stimulation have been studied in skin. [START_REF] Defrin | Interactions between spatial summation, 2-point discrimination and habituation of heat pain[END_REF] A shorter stimulus duration has been shown to reduce nociceptor activation times and produce a stronger spatial-temporal summation at central synapses. [START_REF] Iannetti | A delta nociceptor response to laser stimuli: selective effect of stimulus duration on skin temperature, brain potentials and pain perception[END_REF] The pain scores reported by Al-Hussainey and co-workers were better than our NPS scores for 20ms and 100ms photocoagulation, although far less laser burns were used in their study and a single eye received two different types of laser that would confound the overall perception of pain by the patient. [START_REF] Al-Hussainy | Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times[END_REF] The mean age of patients and the laser power parameters for the 100ms group was higher compared to our study. We found no correlation between NPS and NHS scores and age or sex. Higher fluence laser in an older age-group with less retinal pigmentation may explain the differences between pain scores in both studies. The pain responses demonstrated in our study are more closely related to clinical practice, as 1500 burn PRP is within ETDRS recommendations for treatment.

Previous PDR studies have not as yet studied physiological, psychological, and spatialtemporal effects of retinal laser photocoagulation at different pulse durations.

Our study did not demonstrate any significant effects or differences between 20ms and 100ms PRP on physiological aspects of pain. Depressed mood and anxiety was reported in 10-20% of patients in both laser groups, and photopsia was problematic in 25% cases following 100ms-PRP. At home, patients reported no significant impairment of reading and television tasks, with similar and equivalent responses when either navigating at night or driving. Our small sample size may explain the difficulty in associating any physiological responses with different PRP techniques.

The main limitations of our study include the absence of conventional argon laser (514nm) PRP as a comparative group. However the Pascal® system (532nm) allows 100ms burns in single-spot mode, and we used a single laser system with standard illumination and optical apparatus for both arms of the study. It may be questionable as to the exact source of pain during the laser treatments. Patients reported any discomfort from the laser illumination source at the onset of laser titration before the PRP had commenced, and light intensity was adjusted accordingly until patients were comfortable and able to fixate with the fellow eye during the PRP. There were no reports of increased discomfort or pain associated with the multiple aiming beams of the multi-spot Pascal® raster patterns. Pre-laser topical anaesthesia minimised any surface discomfort from the contact lens, and pain related to contact lens was reported in only 20% of all eyes.

Multi-spot 20ms-PRP laser under topical 0.4% oxybuprocaine is associated with significantly lower levels of anxiety, headache, pain, and photophobia compared to single-spot 100ms-PRP treatment. This may be explained by a combination of lower fluence, shorter-pulse duration, and spatial summation of laser nociception. "The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms/)."

Legends:

  Competing interests: GR Marcellino is an employee of Optimedica Corporation. PE Stanga has received financial support from Optimedica Corporation Licence for Publication.

Figure 1 :Figure 2 :

 12 Figure 1: Consort flow chart of study

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4:

  Photopsia 2 5

Table 1 .

 1 Study Eye Major Inclusion and Exclusion Criteria Mean CRT of less than 300 microns as measured by OCT scans with absence of intra and/or subretinal fluid • Adequate pupil dilatation and clear media to perform laser photocoagulation, digital photography and OCT scans • Ability to perform accurate Humphrey visual field test • If both eyes are eligible, then both eyes will be randomized as per protocol and treated independently

	Inclusion Criteria
	• Older than 18 years of age
	• Male or female patients with diabetes mellitus type I or type 2 who meet the WHO or
	ADA criteria for diabetes
	• ETDRS visual acuity between 35 and 85 letters (Snellen equivalent of 6/60 or better).
	• Newly diagnosed PDR
	• Exclusion Criteria
	• Recent (last 6 months) or ongoing poor glycemic control. HbA1C greater than
	10.0mg/dL
	• Uncontrolled hypertension. Blood pressure greater or equal to 180/110 mmHg
	• History of chronic renal failure or renal transplant for diabetic nephropathy
	• Lens opacity/cataract that could influence vision and results
	• Any previous surgical or laser treatment to the study eye or fellow eye
	• Planned YAG peripheral iridotomy
	• Previous laser photocoagulation or macular laser treatment to study eye or fellow eye.
	• History of DME in study or fellow eye
	• Any previous ocular condition that may be associated with a risk of macular oedema
	• Active lid or adnexal infection
	• Previous retinal treatment: laser, drug or surgery
	• Planned intra-ocular surgery within one year
	WHO=World Health Organization; ADA=American Diabetic Association; ETDRS=
	Early Treatment Diabetic Retinopathy Study; PDR=proliferative diabetic retinopathy;
	CRT=retinal thickness within central subfield; OCT=optical coherence tomography;
	HbA1C=glycosylated hemoglobin; YAG= yttrium aluminum garnet; DME=diabetic
	macular edema

Table 2 .

 2 Pascal® Laser Parameters

		Single-Session	Multiple-session
		Group	Group
	Number of sessions	One	Three
		Day 0	Day 0	Day 14	Day 28
	Number of burns	1500	500 per session (total-1500)
	Type of laser	Pascal® (532nm)	Pascal® (532nm)
	Type of laser spot	Pattern-spot	Single-spot
		5x5, 4x4 arrays
	Spot-size	400µm	400µm
	Pulse duration	20ms	100ms
	Laser burn spacing	1.5 burn-widths	1.5 burn-widths
	Laser burn intensity	Grade 2+, 3+ ETDRS	Grade 2+, 3+ ETDRS
		Mild gray-white	Mild gray-white

Table 3 :

 3 Demographics for each group

	Single Session	Multiple Session
	Pascal® 20ms	100ms

Table 4 :

 4 Number of patients reporting side-effects immediately after Laser

	Side-effect	Single Session	Multiple Session
		(n=20)	(n=20)
	Ache	5	9
	Pinprick	5	2
	Sharp	1	3
	Burning	2
	Tingling	1
	Electric shock-like		1
	Intense		1
	Discomfort		1
	Anxiety	4	10
	Nausea		1

Table 5 :

 5 Physiological side-effects during first month following laser treatment

	Side-effect	Single Session	Multiple Session

Acknowledgements: This research has been supported by the Manchester Academic

Health Sciences Centre (MAHSC) and NIHR Manchester Biomedical Research Centre