FRCSEd(Ophth Inez B Y Wong

BSc(Hons Varun D Sukthankar

PhD Mario Cortina- Borja

FRCOphth Ken K Nischal

Incidence of Early Onset Glaucoma after Infant Cataract Extraction With and Without Intraocular Lens Implantation

Keywords:

Aims:

To determine the incidence of glaucoma, with onset within one year after the date of cataract surgery (early onset) performed in the first year of life, with or without intraocular lens (IOL) implantation.

Methods:

A retrospective review of a single surgeon's cohort from 1999 to 2006.

Glaucoma onset risk, comparison of aphakic/ pseudophakic eyes and IOL type were analysed together with microcornea, persistent fetal vasculature (PFV) and age ≤4 weeks at surgery.

Results:

Ninety-eight eyes(62 patients;mean age 2.88 months) were included with 61 eyes(36 patients) aphakic (57 planned and 4 failed implantations), and 37 eyes(26 patients) pseudophakic. At a mean follow-up of 2.51 years,15.3% (12.2% within 1 year) of all eyes, 9.8% of eyes (6.6% within 1 year) in the planned aphakic group, all 4 eyes with failed implantation and 13.5% of the pseudophakic eyes (10.8% within 1 year) developed glaucoma. Glaucoma incidence stratified by absence or presence of IOL showed no statistically significant difference, but eyes in the rigid polymethylmethacrylate group had an increased glaucoma risk compared to the Acrysof® group (p=0.002). Microcornea, PFV and Age ≤4 weeks at surgery were not significant predictors of early onset glaucoma.

Conclusions:

In this single surgeon study of infant cataract surgery only, age ≤4 weeks at surgery was not a predictor of early onset glaucoma. The rate of aphakic and pseudophakic early onset glaucoma was not found to be statistically different but we found a statistically different rate of glaucoma between the two IOL types which needs 4 further evaluation given that this is a retrospective review. Excessive surgical trauma influences incidence of glaucoma.

INTRODUCTION

Despite advances in congenital cataract management, glaucoma after surgery remains a challenging problem. [START_REF] Chrousos | Incidence of chronic glaucoma, retinal detachment and secondary membrane surgery in pediatric aphakic patients[END_REF][START_REF] Simon | Glaucoma after pediatric lensectomy/ vitrectomy[END_REF][START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF][START_REF] Chak | Incidence of and factors associated with glaucoma after surgery for congenital cataract. Findings from the British Congenital Cataract Study[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] Earlier surgery may give the best chance for visual rehabilitation but with a possible increased glaucoma risk. Primary IOL implantation has become increasingly common in infants but studies implying that IOL implantation may be protective against glaucoma, [START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF] are likely flawed by the inclusion of all ages up to 18 years, with an IOL selection bias for the older age group. Multiple surgeons with varied surgical techniques may be a further confounding factor.

Despite numerous studies [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF][START_REF] Chak | Incidence of and factors associated with glaucoma after surgery for congenital cataract. Findings from the British Congenital Cataract Study[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] about aphakic/pseudophakic glaucoma, there is no consistent definition of glaucoma with intraocular pressure (IOP) threshold ranging from more than 21 mmHg to ≥26 mmHg. [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] Secondary ocular changes are only variably included in the diagnostic criteria, [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] while others base their diagnosis on the decision of the ophthalmologist to treat without defining glaucoma. [START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Chak | Incidence of and factors associated with glaucoma after surgery for congenital cataract. Findings from the British Congenital Cataract Study[END_REF] More recently it has been suggested that aphakic glaucoma onset may be earlier than previously suggested. [START_REF] Chak | Incidence of and factors associated with glaucoma after surgery for congenital cataract. Findings from the British Congenital Cataract Study[END_REF] The present study is limited to infants who underwent cataract surgery with or without IOL implantation by a single surgeon at a single centre. The aim is to determine and compare the incidence of early onset glaucoma in these infants.

METHODS

All lensectomies performed in patients 1 year old or younger between 1999 and 2006 by a single surgeon (KKN) at Great Ormond Street Hospital for Children were identified by database search. The study was approved by the Hospital institutional review board and adhered to the tenets of the Declaration of Helsinki. Exclusion criteria include preexisting glaucoma, history of trauma, serious ocular malformation, Lowe syndrome and maternal rubella syndrome. Eyes with persistent fetal vasculature (PFV) were included.

Eyes with microcornea defined as horizontal cornea diameters (HCD) <10mm, or <9.5mm in those less than 4 months of age, at the time of surgery were also included. [START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Wallace | Corneal diameter in childhood aphakic glaucoma[END_REF] Data extraction included patients' age at surgery, associated systemic and ocular anomalies, family history, cataract type, surgical technique, IOL implantation, type of IOL, intra-and post-operative complications. Pre-and post-operative evaluations included visual assessment, IOP, HCD and optic nerve appearance, date of glaucoma onset, length of follow-up, IOP at final follow-up, and type of glaucoma intervention.

IOP was measured at every clinic visit using either Pulsair tonometer (Keeler, Windsor, UK) or Perkins hand-held tonometer (Clement Clarke, London, UK). In cases where this was not possible, examinations under anaesthesia were performed to evaluate for glaucoma.

Glaucoma was defined as a sustained rise in IOP >21mmHg OR one or more ocular signs of glaucoma (progressive optic disc cupping, increase in horizontal corneal diameter, increased myopic shift, and corneal clouding) OR both. If a child needed anaesthetic examination with or without intervention, gonioscopy was performed. Early onset glaucoma was glaucoma detected less than one year after cataract surgery.

Surgical technique

Eyes with microcornea or capsular bag diameter of less than 8mm as measured using preoperative ultrasound were excluded from implantation of IOL. The operations were performed by a single surgeon. In all cases, lensectomy was performed through two corneal incisions using automated aspiration and irrigation, followed by posterior capsulotomy and anterior vitrectomy. In cases where an IOL was implanted, a TIPP capsulorrhexis technique [START_REF] Nischal | Two-incision push-pull capsulorrhexis for pediatric cataract surgery[END_REF][START_REF] Hamada | Five-year experience of the 2-incision push-pull technique for anterior and posterior capsulorrhexis in pediatric cataract surgery[END_REF] was used for both the anterior and posterior capsulotomy.

Biometry was performed at the time of surgery. Either a rigid heparin-surface-modified polymethylmethacrylate (HSM-PMMA) IOL (Pharmacia 812C) or a foldable 3-piece or 1-piece Acrysof® IOL (MA60AC or SA60AT, Alcon laboratories) was implanted. If inthe-bag implantation was not achieved the IOL would be removed and the patient left aphakic. Posterior capsulotomy and anterior vitrectomy was performed via a pars plicata approach after lens implantation except when the axial length was greater than 20mm when the lens was implanted after posterior capsulorrhexis and anterior vitrectomy via an anterior approach. No IOLs were placed in the sulcus.

Statistical analysis

The risk of onset of glaucoma was modeled using Kaplan-Meier estimators, and the log rank test was used to compare the risk of glaucoma in aphakic eyes versus pseudophakic eyes. A multivariable Cox proportional hazards regression model was developed, which tested potential predictors of glaucoma: microcornea, PFV and age at the time of surgery.

Model selection was based on likelihood ratio tests; these allowed determination of the relative importance of the predictors. Comparison of mean follow-up was performed using the Wilcoxon rank test. In the pseudophakic group, Fisher's Exact Test was used to compare the risk of glaucoma in the HSM-PMMA versus Acrysof® groups. All calculations were performed in R version 2.5.0 in a windows environment. [START_REF]R: A Language and Environment for Statistical Computing[END_REF] While we were interested in early onset glaucoma, statistical analysis was performed on all cases of glaucoma since this cohort includes cases where risk of glaucoma is thought to be highest (i.e. ≤1 year). [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Walton | Pediatric aphakic glaucoma: a study of 65 patients[END_REF]

RESULTS

A total of 98 eyes of 62 patients with a mean age of 2.88 months (range 0.33 to 12.73) at the time of surgery were included. Sixty-one eyes of 36 patients were aphakic and 37 of 26 patients received primary intraocular lens implantation. In the aphakic group the mean age was 2.00 months (range 0.33 to 7.70), and in the pseudophakic group 4.31 months (range 0.73 to 12.73). Mean follow up was 2.51 years for the entire cohort (range 0.1 to 7.84), and similar in both the aphakes (mean 2.61yrs, range 0.1 to 7.84) and pseudophakes (mean 2.32, range 0.1 to 7.24) groups (Wilcoxon test p = 0.4547).

In 4 cases an implant was placed in the bag, only to be removed because of posterior IOL migration. Since no IOL was placed in the sulcus these lenses were removed often with much iris manipulation. All 4 cases developed glaucoma within 14 weeks; mean onset at 1.34 months (range 0.33 to 3.27 months). This group is included in the statistical analysis of patients who had planned lensectomy without IOL but it should be noted that the cases within this group represent an unusual cohort.

The overall incidence of glaucoma was 15.3% (15 in 98 eyes, 14 patients), with 12.2% occurring within the first year. In the aphakes, 10 out of 61 eyes (16.4%, 95% CI 0.092, 27.6) developed glaucoma at a mean of 5.03 months (range 0.1 to 27.1) (13.1% within 1 year), whilst in the pseudophakes, 5 out of 37 eyes (13.5%, 95% CI 0.059, 28.0) developed glaucoma at a mean of 5.56 months (range 0.67 to 14.70) (10.8% within 1 year); there was no significant difference between both groups (Fisher's Exact Test p = 0.7793). Kaplan-Meier estimates of glaucoma incidence stratified by absence or presence of IOL implantation at the time of lensectomy showed no significant difference (p = 0.752, Hazard ratio 0.84, 95% CI = 0.286, 2.47) (Figure 1). Exclusion of the 4 eyes with explantation the mean onset of glaucoma in the aphakes increases from 5.03 months to 7.49 months.

After adjusting for age at the time of surgery, Kaplan-Meier analysis of glaucoma incidence stratified by absence or presence of IOL implantation at surgery again showed no significant difference ( p = 0.99, Hazards ratio 1.01, 95% CI 0.35, 2.9) if the IOL group was considered homogenous. However, of the 37 eyes that had primary IOL implantation, 12 had rigid HSM-PMMA IOL and 25 had foldable acrylic IOLs (MA60AC, 24 eyes and SA60AT, 1 eye). All 5 pseudophakic eyes which developed glaucoma had rigid HSM-PMMA IOLs and none of the eyes which had foldable acrylic IOLs were similarly affected. Comparison of HSM-PMMA vs acrylic IOLs groups using Fisher's Exact Test showed a statistically significant increased risk of glaucoma in the HSM-PMMA group (p = 0.002, Adjusted odds ratio: 37.4, 95% CI 2.46, Infinity). (See Figure 2). Age of 4 weeks or less at the time of surgery was not found to be a significant predictor of glaucoma ( p = 0.064, Hazards ratio 0.32, 95% CI 0.096, 1.07). In the aphakes, 9 eyes were considered to have microcornea, and 1 of these eyes developed glaucoma. In the pseudophakes, our selection criteria for primary implantation excluded eyes with microcornea. Ten patients had cataracts associated with PFV (6 in the aphakic group, and 4 in the pseudophakic group), and of these, 2 in the aphakic group and 1 in the pseudophakic group developed glaucoma. However, both microcornea and PFV were not found to be significant predictors of glaucoma. (See Table 1 Five pseudophakes developed glaucoma. Only one was controlled on medication alone. The remaining 4 eyes had diode cyclophotocoagulation (mean 2.25, range 2 to 3).

One eye went onto a trabeculectomy and subsequently required an Ahmed tube for IOP control.

In addition, 6 eyes in the aphakic group and 1 eye in the pseudophakic group developed a transient rise in IOP requiring short-term medication in the early postoperative period. All cases resolved completely with no other sign of glaucoma and thus

were not considered to have glaucoma.

Visual axis opacification (VAO) requiring surgical clearance occurred in 15 pseudophakes (40.5%) and 4 aphakes (6.6%). All 5 pseudophakic glaucoma cases had had VAO clearance, but only 1 of the aphakic glaucoma cases.

DISCUSSION

Surgery during the first year of life is thought to increase the risk of glaucoma. [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Walton | Pediatric aphakic glaucoma: a study of 65 patients[END_REF] and therefore this cohort is high risk eyes for development of glaucoma. Previous studies indicate a bimodal aphakic glaucoma onset: [START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF][START_REF]R: A Language and Environment for Statistical Computing[END_REF] with early glaucoma presenting within the first year after lensectomy significantly associated with angle closure, and a late open angle glaucoma occurring much later, [START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF][START_REF]R: A Language and Environment for Statistical Computing[END_REF] but not all these studies were restricted to infants. The aim of our study was to look specifically at "early onset" secondary glaucoma occurring after infant cataract surgery. All our glaucoma cases had an open angle type configuration except for one aphake with pupil block. The mechanism of secondary open angle glaucoma after infant cataract surgery is unknown, but may be related to trabeculitis, release of chemical factors from the vitreous which are toxic to the trabecular meshwork, [START_REF] Gimbel | Results of bilateral cataract extraction with posterior chamber intraocular lens implantation in children[END_REF] or acquired micro-peripheral anterior synechiae. [START_REF] Walton | Pediatric aphakic glaucoma: a study of 65 patients[END_REF] In our cohort all patients who had an implantation which was aborted all developed glaucoma within 14 weeks of surgery. Implantation was aborted after the IOL showed evidence of posterior migration usually through the posterior capsulorhexis. Under such circumstances we have been reluctant to place an IOL in the sulcus because of two reasons: firstly, when an IOL is placed in the ciliary sulcus, studies have shown that the haptics cause chronic inflammatory changes with peripheral anterior synechiae [START_REF] Ozdal | Ultrasound biomicroscopy of pseudophakic eyes with chronic postoperative inflammation[END_REF] and induce iris root/ trabecular meshwork changes. [START_REF] Hato No | Histopathological study of anterior segments of the eye with the intraocular lens in the posterior chamber[END_REF] Secondly, one of the few studies to suggest that the axial growth of the pseudophakic infant eye is arrested/ retarded was one in which the IOLs were all places in the ciliary sulcus of infant eyes. [START_REF] Griener | Effect of age at time of cataract surgery on subsequent axial length growth in infant eyes[END_REF] Since all explanted eyes developed glaucoma we speculate that the protracted surgical trauma may lead to increased evidence of early onset aphakic glaucoma.

Our rate of early onset aphakic glaucoma (excluding the subgroup with IOL explantation) is 6.6% (4 of 61 eyes) which is similar to other recent work, [START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] but these authors reported all their cases to be due to pupil block. Our rate is likely to rise the longer the follow up, but the purposes of this study were to look at early onset glaucoma.

The mean time to glaucoma diagnosis was 5.03 months (range 0.1 to 27.1) in the total aphakic group and 5.56 months (range 0.67 to 14.70) in the pseudophakic group.

Other studies looking at infants cataract surgery reported a later onset generally, but they either use criteria at IOP > 25mmHg [START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Kuhli-Hattenbach | Risk factors for complications after congenital cataract surgery without intraocular lens implantation in the first 18 months of life[END_REF] or base diagnosis on clinicians' decision to treat without further detail [START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF] or do not define any criteria. [START_REF] Lundvall | Primary intraocular lens implantation in infants: Complications and visual results[END_REF] With a relatively low threshold of 21 mmHg, and examination under anaesthesia being routinely performed when IOP measurement was not obtained in the clinic, our study is likely to detect most cases of glaucoma early.

Although some studies have shown that primary IOL implantation may prevent or reduce the incidence of glaucoma, [START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF] we did not find a statistically significant difference in terms of glaucoma-free survival when we compared our aphakic and pseudophakic groups in concordance with other recent work. [START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF] A higher risk of secondary glaucoma has been found when surgery is performed early, with studies using a variable threshold of either 4 weeks, 4,8 9 months, [START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF] or 1 year of age. [START_REF] Chen | Risk factors for the development of aphakic glaucoma after congenital cataract surgery[END_REF] Using a cutoff of 4 weeks, we did not show a statistically significant effect of age on the rate of early onset glaucoma. This may be due to shorter follow-up.

We did not find microcornea in itself to be a significant predictor of glaucoma in our multivariate analysis and this could be explained by selection bias, but it should be noted that the capsular bag diameter was often used to determine whether an implant could be placed or not. Microcornea has been variably shown to be associated with development of aphakic glaucoma. [START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Wallace | Corneal diameter in childhood aphakic glaucoma[END_REF][START_REF] Gimbel | Results of bilateral cataract extraction with posterior chamber intraocular lens implantation in children[END_REF] PFV was not found to be a significant predictor of glaucoma in our series. All cases in our series were performed by a single surgeon using standard surgical techniques, which may overcome some of the compromise in the other studies where multiple surgeons were involved. [START_REF] Vishwanath | Is early surgery for congenital cataract a risk factor for glaucoma?[END_REF][START_REF] Rabiah | Frequency and predictors of glaucoma after pediatric cataract surgery[END_REF][START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF][START_REF] Asrani | Does primary intraocular lens implantation prevent "aphakic" glaucoma in children?[END_REF][START_REF] Lawrence | Glaucoma following cataract surgery in children: surgically modifiable risk factors[END_REF][START_REF] Swamy | Secondary glaucoma after paediatric cataract surgery[END_REF][START_REF] Chak | Incidence of and factors associated with glaucoma after surgery for congenital cataract. Findings from the British Congenital Cataract Study[END_REF] Within the pseudophakic group, we found that eyes with HSM-PMMA IOL had a statistically significantly higher rate of glaucoma than the acrylic IOL group. In fact, none of the eyes in the latter group developed glaucoma during the period of follow-up.

We are puzzled by this finding and again with further follow-up this may change. The retrospective nature of this study may have induced an unforeseen sampling error but nevertheless this finding is present. One-piece and three-piece foldable hydrophobic acrylic lenses have superseded the rigid one piece IOLs. [START_REF] Rowe | Primary IOL implantation in children: a risk analysis of foldable acrylic v HSM-PMMA lenses[END_REF][START_REF] Wilson | Acrysof acrylic intraocular lens implantation in children: clinical indications of biocompatibility[END_REF][START_REF] Trivedi | Single-piece acrylic intraocular lens implantation in children[END_REF][START_REF] Nihalani | Single-piece Acrysof intraocular lens implantation in children with congenital and developmental cataract[END_REF] The mean capsular bag diameter is about 7.0 to 7.5 mm at birth and increases to about 9.0 to 9.5 mm after 2 years. [START_REF] Wilson | Intraocular lenses for pediatric implantation: biomaterials, designs, and sizing[END_REF] Comparison of the amount of capsulorhexis ovaling and capsular bag stretch produced by various IOLs implanted in pediatric human cadaveric eyes younger than 2 years found the greatest amount of capsular stretch/ ovaling for the rigid HSM-PMMA IOL. [START_REF] Pandey | Capsulorhexis ovaling and capsular bag stretch after rigid and foldable intraocular lens implantation[END_REF] We speculate that the rigid HSM-PMMA IOL vaults in order to find its resting position in the small infant capsular bag resulting in either forward optic vaulting against the pupil causing pupil block or backward vaulting causing the haptics to push against the iris root. Since none of the pseudophakic cases had pupil block we suspect the haptics caused the iris root to be pushed forward with a possible secondary trabeculitis. While rigid single piece 12mm diameter HSM-PMMA lenses are usually no longer used in the developed countries, they are used in developing countries due to cost pressures. We advise caution when using 12mm diameter rigid IOLs in infant cataract surgery despite the limitations of our retrospective study.

Trivedi reported that for patients who underwent surgery during the first 4.5 months of their life (using hydrophobic acrylic foldable IOLs), the glaucoma incidence was 24.4% in children with pseudophakic eyes and 19% in age-matched children with aphakic eyes [START_REF] Trivedi | Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation[END_REF] . As there is no clarification regarding where the lens is placed (sulcus versus bag), it is possible that the higher incidence of glaucoma in the pseudophakic group may be related to sulcus placed implants based on the effects of sulcus placement described above.

Our study is limited in that it is a retrospective study with relatively short followup but we were specifically looking at early onset glaucoma. We have included eyes with follow-up as short as 0.1 years because we have eyes that developed glaucoma that early.

However, it is one of the largest series of cataract surgery in infants under one year old performed by a single surgeon with identical surgical technique.

Table 1 .

 1 ). Multivariate Cox Proportional Hazards Ratio of Potential Predictors of Glaucoma

	Risk factor	Hazards Ratio 95% CI	P value
	Age at surgery (≤ 4 weeks)	0.319	(0.096, 1.07)	0.064
	Microcornea	0.469	(0.06, 3.68)	0.470
	Persistent Fetal Vasculature (PFV) 2.52	(0.49, 13.12)	0.27

Of the 10 eyes with aphakic glaucoma, one eye developed pupil block glaucoma at 5 weeks post-operatively which responded to anterior vitrectomy. This eye, however, developed glaucoma 3 years later. Two eyes were on medications alone (1 to 2), but 8 eyes required further procedures for IOP control. These included diode cyclophotocoagulation and goniotomy, but two eyes had Ahmed tubes, one of whom underwent trabeculectomy prior to tube insertion.

Competing Interest: None declared. pediatric human eyes. J Cataract Refract Surg 2004;30:2183-91.

LEGENDS


Phillip Jonathan Buckhurst

Prof James Stuart Wolffsohn
email: j.s.w.wolffsohn@aston.ac.uk.

Shehzad Sunil Shah

Anjam Naroo

Nicholas Leon

Emma Julie Davies

Berrow

A NEW OPTICAL LOW COHERENCE REFLECTOMETRY DEVICE FOR OCULAR BIOMETRY IN CATARACT PATIENTS

Keywords: biometry, intraocular lens, cataract, optical low coherence reflectometry, partial coherence interferometry Word Count: 2,350

Background: A new commercially available optical low coherence reflectometry device (Lenstar, Haag-Streit or Allegro Biograph, Wavelight) provides high-resolution non-contact measurements of ocular biometry. The study evaluates the validity and repeatability of these measurements compared to current clinical instrumentation. Method: Measurements were taken with the LenStar and IOLMaster on 112 patients aged 41-96 years listed for cataract surgery. A subgroup of 21 patients also had A-scan applanation ultrasonography (OcuScan) performed. Inter-session repeatability of the LenStar measurements was assessed on 32 patients

Results: LenStar measures of: White-to-white were similar to the IOLMaster (average difference 0.06±0.03D; p=0.305); Corneal curvature were similar to the IOLMaster (average difference -0.04±0.20D; p=0.240); Anterior Chamber Depth were significantly longer than the IOLMaster (by 0.10±0.40mm) and ultrasound (by 0.32±0.62mm; p<0.001); Crystalline Lens Thickness were similar to ultrasound (difference 0.16±0.83mm, p=0.382); Axial Length were significantly longer than the IOLMaster (by 0.01±0.02mm), but shorter than ultrasound (by 0.14±0.15mm; p<0.001). The LensStar was unable to take measurements due to dense media opacities in a similar number of patients to the IOLMaster (9-10%). The LenStar biometric measurements were found to be highly repeatable (variability ≤2% average value).

Conclusions: Although there were some statistical differences between ocular biometry measurements between the LenStar and current clinical instruments, they were not clinically significant. LenStar measures were highly repeatable and the instrument easy to use.

INTRODUCTION

Accurate measurement of ocular biometry is critical to providing optimum refractive outcomes post cataract surgery. [START_REF] Norrby | Sources of error in intraocular lens power calculation[END_REF] Ultrasound is the traditional technique for measuring anterior chamber depth (ACD) and axial length (AL), but is generally limited to a resolution of about ± 0.15 mm. [START_REF] Butcher | The reproducibility of biometry and keratometry measurements[END_REF][START_REF] Raj | Measurement of axial length in the calculation of intraocular lens power[END_REF] Partial coherence interferometry has subsequently been developed as an ocular biometry technique. [START_REF] Hitzenberger | Optical measurement of the axial eye length by laser Doppler interferometry[END_REF][START_REF] Hitzenberger | Measurement of the axial length of cataract eyes by laser Doppler interferometry[END_REF] Since the advent of the first commercial device in 2001 (IOLMaster, Carl Zeiss Jena GmbH), this has become the technique of choice for ocular biometry. Its popularity is due to its non-contact nature, hence avoiding the risk of corneal abrasion and/or contamination, and due to its significantly higher resolution measures of axial length (about ± 0.02 mm; equivalent to 0.05 D). [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] It has been shown to be accurate and repeatable in both cataract biometry assessment [START_REF] Packer | Immersion A-scan compared with partial coherence interferometry -Outcomes analysis[END_REF][START_REF] Nemeth | Optical and ultrasound measurement of axial length and anterior chamber depth for intraocular lens power calculation[END_REF] and in the study of refractive error. [START_REF] Vogel | Reproducibility of optical biometry using partial coherence interferometry -Intraobserver and interobserver reliability[END_REF][START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF] The IOLMaster thus improved the refractive outcome results of cataract surgery [START_REF] Eleftheriadis | IOLMaster biometry: refractive results of 100 consecutive cases[END_REF][START_REF] Rose | Comparison of the Zeiss IOLMaster and applanation A-scan ultrasound: biometry for intraocular lens calculation[END_REF] and by 2002 was used in over a third of hospital eye units in the UK. [START_REF] Gale | National biometry audit[END_REF] However, the IOLMaster only uses partial coherence interferometry to measure AL; corneal curvature, horizontal iris width (white-to-white) and ACD is assessed with imaging techniques and there is no assessment of corneal, crystalline lens or retinal thickness. [START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF] Each of the IOLMaster's three assessments also requires realignment of the device with the visual axis of the eye. It fails to measure in up to 20% of eyes with dense opacities and macular disease, [START_REF] Nemeth | Optical and ultrasound measurement of axial length and anterior chamber depth for intraocular lens power calculation[END_REF][START_REF] Tehrani | Evaluation of the practicality of optical biometry and applanation ultrasound in 253 eyes[END_REF][START_REF] Freeman | The impact of cataract severity on measurement acquisition with the IOLMaster[END_REF] although this can be reduced to less than 10% with more advanced analysis of the interference waveform. [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] Ultrasound is only prevented from measurement in eyes filled with silicone oil, but partial coherence interferometry is not. [START_REF] Tehrani | Evaluation of the practicality of optical biometry and applanation ultrasound in 253 eyes[END_REF][START_REF] Parravano | Reliability of the IOLMaster in axial length evaluation in silicone oil-filled eyes[END_REF] A new ocular biometry device jointly developed by Haag-Streit (LenStar LS900, Haag-Streit Koeniz, Switzerland) and Wavelight (Allegro Biograph, Wavelight, Erlangen, Germany), is now commercially-available. It uses optical low coherence reflectometry to measure corneal thickness, ACD, crystalline or intraocular lens thickness as well as AL. The technique was developed in the late 1980's for reflection measurement in telecommunication devices with micrometer resolution and first applied to in-vivo biological tissue (the eye) by Fercher and colleagues. [START_REF] Fercher | Eye-length measurement by interferometry with partial coherent light[END_REF] The LenStar also assesses central corneal curvature, the horizontal iris width (white-to-white), pupil size, and pupil and visual axis decentration by image analysis, without the need for realignment.

The study evaluates the repeatability of LenStar measurements and its validity when compared to the IOLMaster and A-scan applanation ultrasonograph.

METHODS

One-hundred and twelve patients (36 male and 76 female), with a mean age of 76.4 ± 9.1 years (range from 41 to 96 years, median 77 years) listed for cataract surgery participated in the study. the purpose of the study was explained and informed consent given. All measurements were performed on one eye by a single practitioner for each of the instruments

The study was approved by the National Research Ethics Committee and conformed to the Declaration of Helsinki (2008).

The LensStar, like the IOLMaster, uses the effect of time domain interferometric or coherent superposition of light waves to measure ocular lengths of the eye in a similar technique to one-dimensional optical coherence tomography. The IOLMaster uses a diode laser whereas the LenStar uses a superluminescent diode with a Gaussian shaped spectrum which allows a higher axial resolution; hence the terminology Optical Low Coherence Reflectometry rather than Partial Coherence Interferometry has been coined.

The LenStar was focused and aligned using the image of the eye on the computer monitor while the patient fixated on a flashing red light. The eyes were in focus when the instrument head was approximately 6.8 cm away from the patient's eyes. Patients were asked to perform a complete blink just before measurements were taken in order to spread an optically-smooth tear film over the cornea. The instrument takes 16 consecutive scans per measurement without the need for realignment, and 5 measurements were taken to test intra-session repeatability (as recommended). The device uses optical low coherence reflectometry to measure corneal thickness, ACD, crystalline or intraocular lens thickness and AL using the 820 µm superluminescent diode. The retinal thickness can also be determined from the scans, but this requires subjective alignment of a cursor and was not assessed in this study. It also uses 950 µm light to assess by image analysis; central corneal topography using two rings of diameter 1.65 mm and 2.30 mm (for an eye of radius 7.8mm) of 16 light spot each, reflected off the air / tear interface; the horizontal iris width (white-to-white) by fitting the best circle with the lowest error square to the detected edge; pupil size using the same method; and calculated pupil and visual axis decentration with respect to the centre of the cornea as circumscribed by the limbus.

The IOLMaster, running Advanced Technology version 5 software, [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] was used to assess the same eyes being focused and aligned using the image of the eye on the computer monitor while the patient viewed the instrument's internal illuminated targets. The eyes were in focus when the instrument head was approximately 5.5 cm away from the patient. Patients were asked to perform a complete blink just before measurements were taken in order to spread an optically-smooth tear film over the cornea. AL was measured by partial coherence interferometry (laser diode infrared light of wavelength 780 µm), ACD through image analysis of the distance between the anterior corneal pole and the anterior surface of the crystalline lens illuminated by an optic section, and corneal curvature by image analysis of the distance between three opposite pairs of light spots, arranged in a 2.3 mm diameter hexagonal pattern from the air / tear film interface. [START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF] Five separate measurements were averaged for both AL and corneal curvature, whereas a single shot automatically generated and averaged 5 measures of ACD.

In a subgroup of 21 patients (5 male and 16 female), with a mean age of 78.1 ± 8.1years (range from 70 to 90 years, median 77.5 years) A-scan applanation ultrasound (OcuScan, Alcon Surgical, Irvine, California, USA) was also performed. The A-scan applanation device calculated ACD, crystalline lens thickness and AL from the time taken for ultrasound waves to reflect back to its receiver from an optical surface. [START_REF] Storey | Ultrasound-a research tool in the study of accommodation[END_REF] One drop of topical anaesthetic, benoxinate HCl 0.4 % (Minims ® , Chauvin Pharmaceuticals Ltd, Surrey, UK), was instilled in the patient's eye 2 minutes before ultrasound measurement. Care was taken in aligning the transducer probe along the optical axis and to exert minimal corneal pressure. Ten measurements were taken for each eye and the mean calculated.

The inter-session repeatability of the LenStar was examined by repeating the measurement again in a second session on the same day on 32 of the patients (9 male and 23 female), with a mean age of 73.7 ± 9.3years (range from 41 to 87 years, median 74.5 years).

Statistical Analysis

The bias between measures (the mean difference and 95% confidence interval) were calculated and presented graphically. [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] The level of agreement between biometry measures was tested using the Pearson's Product Moment Correlation Coefficient. Comparison between measures were performed using a paired 2-tailed t-tests. Corneal curvatures were analysed in the steepest and flattest meridian in dioptres, using the refractive index 1.332. As the IOLMaster and ultrasonography determine ACD from the front corneal surface, the corneal thickness calculated by the LenStar was added to its anterior chamber measurement from the back surface of the cornea for comparison.

Results

The average, 95% confidence interval and range of each of the parameters assessed by the LenStar and IOLMaster in this patient population are presented in table 1. Coherence interferometry measurements failed in 10 patients with dense cataract with the LenStar. The IOLMaster could not take partial coherence interferometry measures in these patients and one additional patient. A comparison of the difference between the LenStar and IOLMaster or ultrasound measures for each individual patient compared to the average was plotted for each biometry component. White-to-white corneal measurement was similar as assessed by the LenStar compared to the IOLMaster (Table 2; Figure 1). The LenStar could be expected to read as much as 0.72 mm above to below 0.60 mm the IOLMaster for white-to-white diameter.

Instrument

Corneal curvature measures assessed by the LenStar, were similar to those determined with the IOLMaster (Table 2; Figure 2). The LenStar could be expected to read as much as 0.58 D above to 0.68 D below the IOLMaster for corneal curvature. ACD, as measured by the LenStar was significantly greater than IOLMaster and ultrasound assessment (Table 2; Figure 3). However, there was no apparent bias with the magnitude of the ACD. The LenStar could be expected to read as much as 0.88 mm above to 0.68 mm below the IOLMaster and 1.53 mm above to 0.89 mm below applanation ultrasound for ACD. Crystalline lens thickness as measured by the LenStar was similar to that determined by ultrasound (Table 2; Figure 4).

However, the variability was high with the LenStar expected to read as much as 1.79 mm above to 1.46 mm below ultrasound measures for crystalline lens thickness. AL, as measured by the LenStar was only slightly, but statistically greater than IOLMaster. However, the LenStar determined significantly shorter eyes than ultrasound assessment and there was a bias towards a greater disparity with increasing AL (Table 2; Figure 5). The LenStar could be expected to read as much as 0.06 mm above to 0.04 mm below the IOLMaster and 0.16 mm above to 0.44 mm below applanation ultrasound for AL.

--------------Insert Figures 12345about here --------------

Discussion

The study shows that the validity, repeatability and clinical utility of optical low coherence reflectometry for assessing ocular biometry compared to instrumentation currently used in clinical practice. Only 10% of patients couldn't be measured using the LenStar, similar to the proportion found in this and a previous study with the IOLMaster improved waveform algorithm software. [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] In general, measurements of length/thickness were larger as measured by the LenStar compared to the IOLMaster. However the clinical significance of these effects are minor with the 0.01mm difference in axial length equating to <0.03 D. [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] The greater variability when the device was compared to applanation ultrasonography will be in part due to the lower resolution of this technique [START_REF] Butcher | The reproducibility of biometry and keratometry measurements[END_REF][START_REF] Raj | Measurement of axial length in the calculation of intraocular lens power[END_REF] and because laser light is reflected from the retinal pigment epithelium, in contrast to ultrasound waves which are reflected from the internal limiting membrane. [START_REF] Storey | Ultrasound-a research tool in the study of accommodation[END_REF] A compensation to more closely reflect ultrasound values can be selected in the LenStar software. The IOLMaster does not use coherent interferometry to measure ACD, instead image analysing the distance between the anterior surface of the cornea and crystalline lens when illuminated by an optical section with a 0.7 mm width slit beam of light through the anterior segment of the eye at an angle of 38° to the visual axis [START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF]. The LenStar detects the anterior and posterior corneal, and anterior crystalline lens peaks in the optical low coherence reflectometry waveform to measure the anterior chamber depth and corneal thickness, which were combined for comparison with the IOLMaster result. The shorter ACD measured by ultrasonography compared to the IOLMaster has previous been reported. [START_REF] Reddy | Comparative estimation of anterior chamber depth by ultrasonography, Orbscan II, and IOLMaster[END_REF] The LenStar and IOLMaster were found to measure equivalent values for white-to-white and corneal curvature using image analysis. Caution must be taken when using diopric representation of corneal curvature as differences in the refractive index attributed to the Using the recommended intraocular lens power calculation formulae incorporating many of the discussed biometry measures, the difference between the LenStar and IOLMaster was 0.01 ± 0.30 D (96% within 0.5 D) for SRK II, 0.16 ± 0.30 D (87% within 0.5 D) for Hagis and 0.04 ± 0.24D (95% within 0.5D) for Hoffer Q. [START_REF] Hill | Evaluation of a new IOLMaster algorithm to measure axial length[END_REF] Hence despite some statistical differences between ocular biometry measurements between the LenStar and current clinical instruments, these were not considered to be clinically significant.

The coefficient of repeatability for intra-and inter-session repeatability using the LenStar are impressive (≤2% of the average value for each biometric measure) and at least comparable with the IOLMaster [START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF][START_REF] Lam | The repeatability and accuracy of axial length and anterior chamber depth measurements from the IOLMaster[END_REF] and ultrasound. [START_REF] Butcher | The reproducibility of biometry and keratometry measurements[END_REF][START_REF] Raj | Measurement of axial length in the calculation of intraocular lens power[END_REF] As expected, using the average of repeated measurements decreases the variability and this can be further improved by excluding the most divergent of the results as allowed by the functionality of the LenStar software.

Compared to currently used clinical instrumentation, the LenStar provides a comprehensive range of ocular biometry measurements required by newer, more accurate intraocular lens power calculation formulae. [START_REF] Fenzl | Refractive and visual outcome of hyperopic cataract cases operated on before and after implementation of the Holladay II formula[END_REF] In addition it allows measurements such as corneal thickness (including the functionality of measurement while the patient views internal off-axis illuminated targets at 2mm and 2.7mm eccentricity separated by 22.5°), retinal thickness and the decentration between the visual axis and the centre of the cornea. Some of these measures may improve the accuracy of optimal intraocular lens power prediction or be useful in assessing the development of refractive error. [START_REF] Santodomingo-Rubido | A new non-contact optical device for ocular biometry[END_REF] It is therefore envisaged that the LenStar will be well received in both the clinical and research environment due to its high resolution, good validity and repeatability compared to currently used instrumentation, single alignment requirement and non-contact measurement.

  cornea between the instruments (n=1.3375 [IOLMaster] and n=1.332 [LenStar]) would result in a clinically significant difference in average curvature for both medians of 0.76 ± 0.21 D (p < 0.001) in this study population. The LenStar measures of crystalline lens thickness were not correlated to those recorded by ultrasonography. The larger intra-session variability (±0.33 versus ±0.09 mm) and range of values (2.83-5.06 versus 3.72-5.38 mm) with ultrasound compared to the LenStar suggests optical low coherence reflectometry may be the better technique to assess crystalline lens thickness.

Figure 1

 1 Figure 1White-to-white: difference between LenStar and IOLMaster. Solid line denotes

Figure 2

 2 Figure 2Corneal Curvature: difference between LenStar and IOLMaster in the flattest

Figure 3 Anterior

 3 Figure 3Anterior Chamber Depth: difference between LenStar and IOLMaster / A-

Figure 4

 4 Figure 4Crystalline lens thickness: difference between LenStar and A-Scan

Figure 5

 5 Figure 5Axial Length: difference between LenStar and IOLMaster / A-Scan

Table 1 :

 1 Average ± 95% confidence interval and [range] of biometry measurements as assessed by the LenStar and IOLMaster. Failed measurement refers to coherence interferometry measures. n=112

	LenStar	IOLMaster

Table 2 :

 2 Average ± 95% confidence interval, significance (p) and correlation (r) of IOLMaster (n=101) and Ultrasonography (n=21) with the LenStar biometry measures.LenStar intra-session and inter-session variability was small, with inter-session variability in the average reading being consistently smaller than the intra-session variability between measures for optical low coherence interferometry and corneal curvature measures. This

	Instrument
		IOLMaster	Ultrasound
	Biometry
	White-to-white diameter (mm)	0.06±0.33
		p=0.305
		r =0.74
	Corneal curvature (D)	-0.03±0.31
	flat meridian	p=0.308
		r =0.98
	Corneal curvature (D)	-0.05±0.32
	steep median	p=0.130
		r = 0.97
	Anterior Chamber Depth (mm)	0.10±0.40	0.32±0.62
		p=0.014	p=0.028
		r = 0.68	r=0.36
	Crystalline lens thickness (mm)		0.16±0.83
			p=0.382
			r=0.03
	Axial Length (mm)	0.01±0.02	-0.14±0.15
		p<0.001	p<0.001
		r = 0.99	r=0.99

standard deviation of repeated measures with the LenStar.

Acknowledgements:

The LenStar was loaned to the authors by Haag Streit for the duration of the study.

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in British Journal of Ophthalmology and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence http://bjo.bmj.com/ifora/licence.pdf

Competing Interests:

None declared


M.D Simone Kellner
email: simonekellner@mac.com

Silke Weinitz

Ulrich Kellner

Spectral domain optical coherence tomography detects early stages of chloroquine retinopathy similar to multifocal electroretinography, fundus autofluorescence and near-infrared autofluorescence

Keywords: retina, electrophysiology, imaging, drugs Financial relationship: none for all chloroquine, FAF: fundus autofluorescence, HCQ: hydroxychloroquine, mfERG: multifocal electroretinography, NIA: near-infrared fundus autofluorescence, sdOCT: spectral domain optical coherence tomography

Aims: To compare spectral domain optical coherence tomography (sdOCT) to melanin-related near-infrared fundus autofluorescence (NIA, excitation 787nm, emission >800nm), lipofuscin-related fundus autofluorescence (FAF, excitation 488nm, emission >500nm) and multifocal electroretinography (mfERG) in patients with long-term chloroquin (CQ) treatment.

Methods: Eight patients with 5.5-22 years of CQ treatment underwent clinical examination, mfERG recording, FAF and NIA imaging using a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph 2) and sdOCT imaging (Spectralis OCT Heidelberg Retina Angiograph).

Results:

In three patients all test results were normal after 5.5-16 years of CQ treatment. Five patients presented with variably progressed CQ retinopathy (10-22 years of treatment) and abnormalities in all tests. In the mildest case, pericentral reduction of mfERG amplitudes corresponded to increased pericentral FAF, reduced pericentral NIA and pericentral interruption of the photoreceptor inner/outer segment junction in the sdOCT. In all sdOCT scans outer nuclear layer thickness was reduced. More severe cases showed preserved subfoveal photoreceptors and function with marked changes in all examinations towards the periphery. The most severe case presented with additional loss of subfoveal photoreceptors.

Conclusion: MfERG, FAF, NIA and sdOCT detect early stages of CQ retinopathy.

Loss of outer nuclear layer thickness might be the earliest indicator of CQ retinopathy.

INTRODUCTION

Chloroquine (CQ) and hydroxychloroquine (HCQ) play an important role in the treatment of systemic autoimmune disorders due to their limited side effects. Their major disadvantage is the induction of an irreversible retinal degeneration, which may lead to severe loss of visual fields and visual acuity and subsequent loss of drivers license and occupation. Although the risk of retinal degeneration increases with the duration of treatment, recent findings show that many patients may take CQ or HCQ for many years without problems, whereas few patients develop retinal photoreceptor dysfunction at very low cumulative doses [START_REF] Ruther | Variabilitat der retinotoxischen Gesamtdosis Chloroquin/Hydroxychloroquin[END_REF]. The reason for this variability remains to be established. In animal experiments earliest abnormalities were detected in retinal ganglion cells, but in both animal experiments and histology of human retinas paracentral photoreceptors showed the most severe damage. [START_REF] Bernstein | The Pathology of Chloroquine Retinopathy[END_REF][START_REF] Wetterholm | Histopathology of Chloroquine Retinal Toxicity[END_REF][START_REF] Rosenthal | Chloroquine retinopathy in the rhesus monkey[END_REF] In recent years, retinal dysfunction or retinal structural abnormalities have been identified sometimes prior to ophthalmoscopically visible alterations using multifocal electroretinography (mfERG), [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF][START_REF] Penrose | Multifocal electroretinography evaluation for early detection of retinal dysfunction in patients taking hydroxychloroquine[END_REF][START_REF] Lai | Multifocal electroretinographic changes in patients receiving hydroxychloroquine therapy[END_REF] fundus autofluorescence (FAF), [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF] nearinfrared autofluorescence (NIA), [START_REF] Kellner | Chloroquine retinopathy: lipofuscinand melanin-related fundus autofluorescence, optical coherence tomography and multifocal electroretinography[END_REF] time domain optical coherence tomography (OCT) [START_REF] Kellner | Chloroquine retinopathy: lipofuscinand melanin-related fundus autofluorescence, optical coherence tomography and multifocal electroretinography[END_REF] and a prototype of high speed ultra-high-resolution OCT. [START_REF] Rodriguez-Padilla | High-speed ultrahigh-resolution optical coherence tomography findings in hydroxychloroquine retinopathy[END_REF] Screening for the onset of CQ/HCQ retinopathy is difficult due to the huge number of patients under treatment, unspecific early symptoms (e.g. reading difficulties) and limited availability and high costs for sophisticated diagnostic methods. In the present study we compare commercially available spectral domain OCT (sdOCT) with mfERG, FAF and NIA in different stages of long-term CQ retinopathy and demonstrate that all methods may detect early stages of CQ retinopathy.

PATIENTS AND METHODS

Eight female patients with long-term CQ treatment examined at the AugenZentrum Siegburg were included in this study. All patients were of normal weight at the time of presentation. Results of previous examinations have been reported [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF][START_REF] Kellner | Chloroquine retinopathy: lipofuscinand melanin-related fundus autofluorescence, optical coherence tomography and multifocal electroretinography[END_REF] in some patients that were asked to return for repeated examination including sdOCT (Table 1). Three patients were asymptomatic undergoing regular screening for CQ therapy, whereas five patients had subjective symptoms of visual dysfunction. In these patients, the diagnosis of CQ retinopathy was established based on patient and family history, ophthalmoscopy, visual field testing and mfERG recording according to the standards of the International Society for Clinical Electrophysiology of Vision as reported previously. [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF][START_REF] Marmor | Standard for clinical electroretinography (2004 update)[END_REF][START_REF] Marmor | Guidelines for basic multifocal electroretinography (mfERG)[END_REF] All patients gave informed consent after detailed explanation about the background of the study. The study was performed in adherence to the tenets of the Declaration of Helsinki and approved by the local ethics committee.

FAF and NIA images were obtained as reported previously using a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph 2, Heidelberg Engineering, Heidelberg, Germany) [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF][START_REF] Kellner | Chloroquine retinopathy: lipofuscinand melanin-related fundus autofluorescence, optical coherence tomography and multifocal electroretinography[END_REF][START_REF] Renner | Morphology and functional characteristics in adult vitelliform macular dystrophy[END_REF][START_REF] Kellner | Lipofuscin-and melanin-related fundus autofluorescence visualize different retinal pigment epithelial alterations in patients with retinitis pigmentosa[END_REF]. Spectral domain OCT (sdOCT) was performed with the Spectralis OCT HRA (Heidelberg Engineering, Heidelberg, Germany) according to the manufacturers guidelines using single line mode as well as with 18 scans form the posterior pole using 3D mode. FAF, NIA and sdOCT images were evaluated in comparison to normal images. MfERG recordings were judged based on age-matched laboratory normal values. [START_REF] Kellner | Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine[END_REF]

RESULTS

Patients without CQ retinopathy

In three patients (#2587, 2596, 2702) with CQ treatment over a period of 6 -16 years (Table 1) the structure of all retinal layers appeared normal in all regions in the sdOCT on both eyes (Fig. 1). FAF and NIA imaging as well as visual acuity, visual fields and the multifocal ERG were also normal bilaterally in all three patients. 1) marked retinal structural abnormalities were detected using sdOCT. These patients had noted subjective signs of CQ retinopathy prior to the first detailed examination using retinal imaging. In the patient with the mildest sdOCT changes (#3108, 22 years of interrupted CQ treatment) parafoveal discontinuity of the photoreceptor inner / outer segment junction was observed (Fig. 1). In the three patients (#2688, 2771, 2858) with more advanced CQ retinopathy the foveal photoreceptors were preserved, whereas towards the periphery the layers of the outer segments, the photoreceptor inner / outer segment junction and the outer nuclear layer could no longer be distinguished (Fig. 1). The alterations were most severe in patient #2858 with 10 years of CQ treatment, less severe in patient #2771 with 20 years of treatment and even lesser in patient #2688 with 14 years of treatment.

In the patient with the most severe sdOCT abnormalities (#3033, 10 years CQ treatment) the foveal and parafoveal region showed severe loss of photoreceptor associated layers. All patients with retinal structural abnormalities in the sdOCT showed also alterations of FAF, NIA and mfERG (Fig. 1). The severity corresponded to the severity of sdOCT changes. An additional sdOCT finding in patient #3108 was the general reduction of outer nuclear layer thickness (Fig. 2).

DISCUSSION

Patients usually tolerate CQ or HCQ treatment well and often have to change to drugs with more frequent side effects when CQ/HCQ therapy has to be discontinued. Therefore it is important to reliably diagnose early stages of CQ/HCQ retinopathy. The present study shows that in our patients with CQ treatment mfERG, FAF, NIA and sdOCT where either normal or all tests showed at least mild abnormalities. In patients with CQ retinopathy the alterations of the photoreceptor layers documented with the sdOCT included loss of photorecptor inner segments indicated by the reduced outer nuclear layer thickness and the interruption or absence of the photoreceptor inner/outer segment junction. These findings are in agreement with a previous report using a research type of high-definition OCT. [START_REF] Rodriguez-Padilla | High-speed ultrahigh-resolution optical coherence tomography findings in hydroxychloroquine retinopathy[END_REF] These structural retinal alterations correspond to photoreceptor dysfunction demonstrated with the mfERG and an increase of the lipofuscin concentration and a decrease of the melanin concentration in the RPE as seen in FAF and NIA images [START_REF] Gibbs | Retinal pigment epithelium defects in humans and mice with mutations in MYO7A: imaging melanosome-specific autofluorescence[END_REF], which have been reported previously. [START_REF] Kellner | Chloroquine retinopathy: lipofuscinand melanin-related fundus autofluorescence, optical coherence tomography and multifocal electroretinography[END_REF] It is of importance to note that in the sdOCT the outer nuclear layer shows the most severe changes. Similar changes could be seen on a previous report of OCT alterations. [START_REF] Rodriguez-Padilla | High-speed ultrahigh-resolution optical coherence tomography findings in hydroxychloroquine retinopathy[END_REF] At present, all of the patients examined in both of these studies who presented with sdOCT abnormalities had already visual problems. It is of interest, that even in the patient with the mildest sdOCT changes the outer nuclear layer thickness was reduced in all scans of the 3D series.

Figure 2 :

 2 Figure 2: Fundus reflectance image and upper sdOCT scan of patient #3108 compared to normal sdOCT. The sdOCT shows reduced outer nuclear layer thickness (black bars indicate the outer nuclear layer) in CQ retinopathy compared to two scans of age-matched normal female eyes in the same area.

Table 1

 1

	Patient ID Normal 2702 1 2587 2596 1 CQ retinopathy 3108 2771 2 2688 1* 2858 2 3033	Age at examination [years] 31 59 50 51 53 60 47 68	Duration CQ [years] 16 6 5.5 22 + 20 14 10 10	Estimated cumulative Dose [g] 1460 548 502 1350 + 1825 1278 912 912	Visual acuity OD / OS 1.0 / 1.0 1.0 / 1.0 1.0 / 1.0 1.0 / 1.0 0.7 / 0.8 1.0 / 1.0 0.5 / 0.5 0.1 / 0.2
	1 Previous findings reported in [5]
	2 Previous findings reported in [8]

+ treatment was partly interrupted in some years * Patient developed CQ retinopathy since the previously reported normal findings Patients with CQ retinopathy In five patients (#2688, 2771, 2858, 3033, 3108) with CQ treatment over a period of 3 -20 years (Table

  Throughout the complete scan series the thickness of the outer nuclear layer was reduced compared to a group of 7 agerelated female probands (e.g. most superior scan: #3108: 46.7 µm (mean ± 1 standard deviation: 59.8 ± 7.1 µm), most inferior scan: #3108: 38.3 µm (51.7 ± 5.2 µm). This is of interest as the most superior and inferior sdOCT scans correspond to retinal areas which appeared normal on ophthalmoscopy, FAF, NIA and showed normal amplitudes in the mfERG.

  These changes were detectable in areas where ophthalmoscopy, FAF and NIA appeared normal and mfERG responses were normal as well. Prospective evaluation of outer nuclear layer thickness during CQ/HCQ treatment might provide even earlier suspicion of the onset of retinal degeneration. Based on this limited comparative series, each of the methods appears to detect already milder changes of CQ retinopathy. Similar changes have been reported in HCQ retinopathy using mfERG, FAF and high resolution OCT, it can be concluded that these methods also facilitate early diagnosis of HCQ retinopathy. This is of importance, because not all diagnostic techniques are easily accessible everywhere. As there is no obvious diagnostic advantage of one of the four methods, each might be used when CQ retinopathy is suspected. Characteristic findings confirm the diagnosis, whereas in cases of doubt the other methods can be used to clarify the diagnosis. Normal mfERG amplitudes, normal FAF distribution with low central FAF intensity, normal NIA distribution with high central NIA intensity and normal structure of retinal layers in the sdOCT. Middle row (patient #3108 with mild CQ retinopathy): Pericentral mfERG amplitude reduction (ring 2), pericentral increase of FAF intensity (arrow), pericentral fine reduction of NIA intensity (arrow) and pericentral interruption of photoreceptor inner /outer segment junction (arrows). Lower row (patient #2688 with advanced CQ retinopathy): Marked mfERG amplitude reduction, broad pericentral area of increased FAF (between arrows), marked reduction of NIA intensity and preservation of subfoveal photoreceptors and loss of pericentral and more peripheral photoreceptor layers in the sdOCT in.

	Legends
	Figure 1: Multifocal ERG (mfERG), fundus autofluorescence (FAF), near-infrared
	autofluorescence (NIA) and central spectral domain OCT (sdOCT) scans in
	different stages of CQ retinopathy. Upper row (patient #2702 with long duration
	CQ therapy without retinopathy):

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 British Journal of Ophthalmology and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence


Dr Matthias Lüke
email: matthias.lueke@uk-sh.de

Kai Januschowski

Julia Lüke

Swaantje Peters

Nick Wirtz

Efdal Yörük

Christoph Lüke

Karl Ulrich Bartz-Schmidt

Salvatore Grisanti

Peter Szurman

The effects of ranibizumab (Lucentis ® ) on retinal function in isolated perfused vertebrate retina

Background: Intraocular ranibizumab (Lucentis ® , Novartis, Basel Switzerland) is the primary choice in the treatment of neovascular age-related macular degeneration (AMD).

VEGF is known to be a survival factor for neuronal cells. Therefore, blockage of all VEGF isoforms by ranibizumab could induce retinal dysfunction.

Methods: Using isolated bovine retinas the electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes while the retinas were perfused with an oxygen pre-incubated nutrient solution. For 45 min ranibizumab was applied at a concentration of 0.2mg/mL and alternatively the solvent carrier without the active agent. The ERG was monitored before, during and after exposure.

Results:

The concentration of 0.2mg/mL ranibizumab induced a not significant b-wave reduction of 22.32 % after exposure (p = 0.13). For the a-wave amplitude only a reduction of 4 % was detected (p = 0.18). The solvent carrier induced no significant reduction of the a-and b-wave amplitudes (p = 0.30 and p = 0.979, respectively).

Conclusion:

In our ex vivo model, the isolated perfused vertebrate retina, ranibizumab has been proven to be a safe compound at the concentrations applied. The stability of the ERGamplitudes rules out a considerable retinal dysfunction after an injection of up to 1mg ranibizumab.

Introduction:

Ranibizumab (Lucentis ® , Novartis, Basel, Switzerland) is the humanized fragment of the murine monoclonal antibody that binds all the active isoforms of the vascular endothelial growth factor (VEGF-A) leading to a containment of the neovascular process, the cause of wet age-related macular degeneration (AMD). [START_REF] Ferrara | Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration[END_REF][START_REF] Lowe | Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo[END_REF] Wet AMD leads to a rapid and irreversible loss in central vision and is one of the leading causes of blindness in the western world. [START_REF] Klein | Prevalence of age-related maculopathy. The Beaver Dam Eye Study[END_REF] The development of the anti-vascular growth factor therapy (anti-VEGF) offered a substantial progress and is currently the primary choice in the treatment of neovascular AMD. [START_REF] Pieramici | Ranibizumab: treatment in patients with neovascular age-related macular degeneration[END_REF] Improvement in visual acuity by 15 or more letters has been observed in 33.8-40.3% of patients treated with ranibizumab in pivotal clinical trials. [START_REF] Brown | Ranibizumab versus verteporfin for neovascular age-related macular degeneration[END_REF][START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF] The MARINA study also could show that ranibizumab-treated patients suffering from minimally classic or occult CNV were more likely to improve in vision-related quality of life through 24 months. [START_REF] Chang | Improved vision-related function after ranibizumab treatment of neovascular age-related macular degeneration: results of a randomized clinical trial[END_REF] On the one hand ranibizumab inhibits the effects of VEGF, angiogenesis and vascular permeability, on the other hand, VEGF plays a central role in the trophic maintenance of the choriocapillaris and in protecting retinal neurons from apoptosis in the eye. [START_REF] Ferrara | Vascular endothelial growth factor: basic science and clinical progress[END_REF] While the intravitreous administration of anti-VEGF agents reduces the severity of systemic adverse events, potential concerns about retinal toxicity remain due to the intravitreal injection and to the long-term maintenance therapy that some patients may require.

Clinical trials showed no evidence for retinal toxicity and a therapy with up to 0.5mg ranibizumab intravitreally was well-tolerated for more than two years. [START_REF] Brown | Ranibizumab versus verteporfin for neovascular age-related macular degeneration[END_REF][START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF] But concerns persist, as the pharmaceutical phial of lucentis contains 0.23 mL of a 10mg/mL ranibizumab solution, which makes an accidental injection of more than 0.5mg ranibizumab possible.

The present study was designed to investigate the effects of ranibizumab on the parameters of the electroretinogram using the isolated perfused vertebrate retina technique, an electrophysiological ex vivo technique for evaluation of the safety profile of ranibizumab. [START_REF] Lüke | The isolated perfused bovine retina-A sensitive tool for pharmacological research on retinal function[END_REF][START_REF] Sickel | Respiratory and electrical responses to light stimulation in the retina of the frog[END_REF] This is the first electrophysiological report about testing the effects of ranibizumab on retinal function.

Materials and Methods:

Materials

Aspartate, glucose and other chemicals were purchased from Merck at pro analysis grade.

Ranibizumab, Lucentis ® , was provided from Novartis (Basel, Switzerland). The solvent carrier was made under sterile conditions at the pharmacy of the University Hospital Tübingen in guidance of Novartis. The solvent carrier and ranibizumab was stored at 4°C.

Methods

Superfused vertebrate retina assay: Bovine eyes were obtained directly post mortem and were transported in darkness in a serum-free standard medium containing 120 mM NaCl, 2 mM KCl, 0.1 mM MgCl 2 , 0.15 mM CaCl 2 , 1.5 mM NaH 2 PO 4 , 13.5 mM Na 2 HPO 4 und 5 mM glucose. The preparation was performed as described previously. [START_REF] Sickel | Respiratory and electrical responses to light stimulation in the retina of the frog[END_REF] The electroretinogram was recorded in the surrounding nutrient medium via two silver/silverchloride electrodes on either side of the retina. The recording chamber containing a piece of retina was placed in an electrically and optically insulated box. The perfusion velocity was controlled by a roller pump and set to 1 mL/min. The temperature was kept constant at 30°C. The perfusing medium was pre-equilibrated and saturated with oxygen. The retina was darkadapted and the electroretinogram was elicited at intervals of five min using a 1-Hz single white xenon flash (xenon tube; Grass Technologies, West Warwick, RI 02893 U.S.A.) for stimulation. The flash intensity was set to 6.3 mlux at the retinal surface using calibrated neutral density filters (Kodak Wratten Filter; Kodak, Stuttgart, Germany).

The duration of light stimulation was 10 microseconds controlled by a timer (Photopic Stimulator PS33 Plus; Grass Technologies, West Warwick, RI 02893 U.S.A). The ERG was filtered and amplified (100-Hz high pass filter, 50-hz notch filter, 100 000 x amplification) using a Grass Amplifier P122 (Grass Technologies, West Warwick, RI 02893 U.S.A). The data were processed and converted with an analog-to-digital data acquisition board (PCI-MIO-16XE-50; National Instruments, Austin, Tex, U.S.A) in a desktop computer (PC compatible).

The retina was superfused with the serum-free nutrient solution and stimulated repeatedly until stable amplitudes were recorded. A concentration of 0.2mg/mL ranibizumab was added to the nutrient solution, and responses were recorded for 45 min. Thereafter, perfusion with standard solution was resumed for another 75 min to observe the b-wave recovery. According to the described procedure one pilot experiment was performed to evaluate the effects of ranibizumab at a concentration of 0.1mg/mL to simulate the impact of the clinical dose, 0.5mg ranibizumab, on the b-wave amplitude. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave (Fig. 1a).

To investigate the effects of ranibizumab on the photoreceptors under scotopic conditions (6.3 mLx flash light intensity), the b-wave was suppressed by adding 1 mM aspartate to the nutrient solution. Under these conditions, the influence of ranibizumab on the photoreceptor potential P III was analyzed. Aspartate is an inhibitor of synaptic transmission at the level of the first retinal synapse and enables the recording of unmasked photoreceptor potential P III by abolishing the b-wave (Fig. 1b). [START_REF] Hanawa | The Effect of Aspartate on the Electroretinogram of the Vertebrate Retina[END_REF] We recorded stable photoreceptor potential P III for 30 minutes. Thereafter, ranibizumab was added in a concentration of 0.2mg/mL to the aspartate containing nutrient solution for 45 minutes. The changes of the a-wave amplitude during the application of ranibizumab were recorded and the recovery was followed up for 75 minutes under perfusion with the aspartate containing nutrient solution.

To test the effects of the solvent carrier on the a-and b-wave amplitude the solvent carrier without the active agent ranibizumab was used as a control. Equivalent amounts of the solvent carrier were applied to the nutrient solution for 45 min under the same conditions as during the testing of the ranibizumab. Subsequently, the a-and b-wave recovery was observed for 75 min.

Data analysis:

The percentage reduction of the a-wave and b-wave amplitude was calculated before and after application of ranibizumab. The a-and b-wave recovery was compared with the a-and b-wave amplitude before application of ranibizumab and the solvent carrier, respectively. For the statistical analysis the software "Origin 6.0" (Microcal) was used.

Significance was estimated by the Student`s t-test and levels of p ≤ 0.05 were considered as statistically significant.

Results:

The perfusion of the isolated bovine retina was performed under stable environmental conditions. Osmotic pressure, temperature, and pO 2 remained unchanged during perfusion. In our experimental setting stable ERG amplitudes were reached within two hours of perfusion.

We showed that the superfused retinal preparations responded constantly to light stimulation in our experimental setup for more than ten hours. [START_REF] Lüke | The isolated perfused bovine retina-A sensitive tool for pharmacological research on retinal function[END_REF] Ranibizumab was effective in the treatment of neovascular AMD at an intravitreal dose of 0.5mg dissolved in 0.05 mL solvent carrier per human eye according to a concentration of 0.1mg/mL. To investigate the safety and the retinal tolerance we tested a higher dose of ranibizumab. According to the volume of the vitreous humour of approximately 5 mL, the averaged intraocular concentration of ranibizumab was calculated to 0.2mg/mL.

The concentration of 0.2mg/mL ranibizumab failed to induce a significant decrease of the bamplitudes of the isolated perfused bovine retinas, although a non-significant b-wave reduction of 22.32 % was found directly after exposure (p = 0.13, Fig. 2A). After exposure the b-wave amplitude recovered completely during the washout reaching the mean value of bwave amplitudes before the application of ranibizumab and no delayed decrease of the b-wave amplitudes could be observed (p = 0.36, Fig. 2A).

In contrast, the same amount of the solvent carrier reduced the b-wave amplitude only about 2.9 % (p = 0.979, Fig. 3A). In addition, the b-wave amplitude remained stable throughout the washout after the exposure with the solvent carrier (p = 0.99, Fig. 3A).

A reduction of the b-wave amplitude alone cannot specify the site of action of any drug on retinal function, which is added to the nutrient solution. Additionally, we investigated the effects of ranibizumab on the photoreceptor potential P III and a-wave amplitude, respectively, using aspartate. [START_REF] Hanawa | The Effect of Aspartate on the Electroretinogram of the Vertebrate Retina[END_REF] After the addition of 1 mM aspartate, the b-wave amplitude was reduced continuously and unmasked photoreceptors potential could be recorded. The concentration of 0.2mg/mL ranibizumab was added to the nutrient solution containing aspartate. After 45 min exposure the a-wave amplitude showed only a slight reduction of 4 % (p = 0.18, Fig. 2B). Similarly, throughout the washout the a-wave amplitude remained unaltered (p = 0.43, Fig. 2B).

In addition, the equal amount of the solvent carrier induced only a not significant reduction of the a-wave amplitude of about 8 % after 45 min exposure (p = 0.30, Fig. 3B). At the washout the a-wave amplitude slightly recovered and the a-wave amplitude remained stable throughout the washout (p = 0.69, Fig. 3B).

One pilot experiment was performed testing the effects of 0.1mg/mL on the b-wave amplitude (Fig. 4). During the exposure a reduction of the b-wave amplitudes could be not detected.

During the washout the b-wave amplitudes were measured unchanged to the b-wave amplitudes before the application of ranibizumab. observation period of two years. Taking into account these considerations, we tested the effects of ranibizumab on the electroretinogram at a higher dose to evaluate the safety profile of intraocular ranibizumab.

Our study revealed no significant decrease of the a-and b-wave amplitude during the exposure of the tested concentration. We found only a not significant reduction of the b-wave amplitude, which indeed seems to be induced by the active agent ranibizumab itself, because the equivalent amount of the solvent carrier did not alter the b-wave. Ranibizumab seems to affect selectively the inner retinal network indicated by the selective b-wave reduction. An alteration of the b-wave amplitude at the concentration of 0.1mg/mL according to the clinical dose of 0.5mg ranibizumab could not be observed. The photoreceptor function itself remained unaltered.

Recently, we tested the effects of bevacizumab (Avastin ® , Roche Pharma, Vienna, Austria), which is the full-length humanized anti-VEGF monoclonal antibody and also proposed in the treatment of wet AMD. For avastin, we found a not significant reduction for the a-and bwave amplitude during the exposure with the threefold dose of bevacizumab (0.8mg/mL according to a dose of 3.75mg), which is recommended to be applied intravitreally [START_REF] Lüke | Effects of bevacizumab on retinal function in isolated vertebrate retina[END_REF]. The observed alterations of the a-and b-wave were most likely induced by the solvent carrier. These findings are not surprising as the solvent carrier of bevacizumab was developed for intravenous cancer therapy and not for intravitreal injections. [START_REF] Horwitz | Bevacizumab plus irnotecan, fluorouracil, and leucovorin for metastatic colorectal cancer[END_REF] In this study, we could demonstrate that the solvent carrier of ranibizumab has no effects on the a-wave as well as on the b-wave amplitude. The origin of the slight dysfunction of the inner retina during the exposure of 0.2mg/mL ranibizumab remained unclear, but was fully reversible after exposure. However, we consider that an injection of the lower and usually injected dose of 0.5mg ranibizumab in the treatment of wet AMD seems to be safe, because a concentration of 0.2mg/mL did not lead to a significant reduction of the b-wave amplitude. In the case of the accidental injection of a higher concentration equivalent to an injected dose of 1mg ranibizumab the effects on retinal function seem to be slight and transient. As higher doses

have not yet been tested in our model, no reliable conclusion can be made on the retinal toxicity of doses higher than 1.0mg ranibizumab.

The clinical experiences and trials confirm our results that an intravitreal injected dose of 0.5mg ranibizumab seems to be safe, which was shown for an observation period of two years with an injection of 0.5mg ranibizumab every four weeks. [START_REF] Brown | Ranibizumab versus verteporfin for neovascular age-related macular degeneration[END_REF][START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF] Clinical experiences with escalating injected doses of 0.3 up to 2mg of ranibizumab showed a mild ocular inflammation as most common post injection adverse event. [16 -18] In these trials the maximum well tolerated dose of ranibizumab was estimated to 0.5mg and a stronger ocular inflammation was observed at higher applied doses. An electrophysiological assessment is missing and was not done in these studies. [16 -18] So far, no electrophysiological data are available for ranibizumab. We think that this additional safety study adds important information to the known clinical data. Especially, in respect to the upcoming of further indications beyond wet AMD the safety profile of ranibizumab should be known since it was shown that ranibizumab was also effective in the treatment of common retinal diseases like the macular oedema induced by diabetes or retinal vein occlusion. [START_REF] Fraser-Bell | Update on treatments for diabetic macular edema[END_REF][START_REF] Spaide | Prospective Study of Intravitreal Ranibizumab as a Treatment for Decreased Visual Acuity Secondary to Central Retinal Vein Occlusion[END_REF] In conclusion, the isolated superfused retina is well defined to evaluate short-term effects on retinal function. Significant short-term toxic effects were not detected at the tested concentration of 0.2mg/mL. This concentration accords to the twofold dose of ranibizumab, which is used in the treatment of wet AMD, and so we could show that ranibizumab seems to be safe also in the case of an accidental injection of more than 0.5mg.

Copyright statement: 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 (http://bjo.bmjjournals.com/misc/ifora/licenceform.shtmL).

Figure 1 :

 1 Figure 1: The ERG from the isolated perfused bovine retina.

Figure 2 :

 2 Figure 2: Effects of Ranibizumab on the ERG of the isolated perfused bovine retina.

Figure 3 :

 3 Figure 3: Effects of the solvent carrier of ranibizumab on the a-and b-wave amplitude

Figure 4 :

 4 Figure 4: Effects of Ranibizumab on the ERG of the isolated perfused bovine retina. The

Fig. 1a

 1a Fig. 1a Lüke et al.

FigFig. 4

 4 Fig. 1b Lüke et al.

This study has been funded by Novartis (Basel, Switzerland).

Discussion:

The ERG is a sensitive parameter of retinal integrity and function. The changes of the ERG in the model of the isolated and perfused bovine retina reflected toxic effects of the drug directly and exclusively on retinal function, in which especially a reduced b-wave indicates an evident dysfunction of retinal neurons. [START_REF] Lüke | The isolated perfused bovine retina-A sensitive tool for pharmacological research on retinal function[END_REF][START_REF] Lüke | Effects of phosphodiesterase type 5 inhibitor sildenafil on retinal function in isolated superfused retina[END_REF] A detailed localization of the effected neuronal layer by the applied drug is enabled by a separate investigation of the photoreceptor function using aspartate. [START_REF] Hanawa | The Effect of Aspartate on the Electroretinogram of the Vertebrate Retina[END_REF] The aim of our study was to evaluate the tolerability of the retina to ranibizumab. We tested a higher concentration according to an intravitreal injection of about 1mg ranibizumab to evaluate the safety profile of ranibizumab also in the case of an accidental injection of a higher dose than 0.5mg ranibizumab, which was the recently evaluated, tested and proposed dose for the treatment of wet AMD. [START_REF] Brown | Ranibizumab versus verteporfin for neovascular age-related macular degeneration[END_REF][START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF] An accidental injection of more than 0.5mg ranibizumab is possible, because the pharmaceutical phial of lucentis contains 0.23 mL.

Deducting the remnants in the phial and a reflux of the solution after injection we calculated a potentially excessive injected amount to 0.1 mL and 1mg ranibizumab, respectively. In 5 mL vitreous humour the applied 1mg ranibizumab would become diluted to the tested concentration of 0.2mg/mL ranibizumab. However, it must also be considered that the spatial distribution inside of the vitreous humour is not uniform and the retina near the area of the injection would be exposed to a higher concentration than the average calculated concentration of 0.1mg/mL, which would be expected after an injection of the usual applied dose of 0.5mg ranibizumab. In addition, the intravitreal concentration of the active agent varies after injection. For example, in monkeys 0.5mg ranibizumab in 0.05 mL leads to a peak vitreous concentration (C max) of 169 µg/mL ranibizumab six hours after intravitreal administration. [START_REF] Gaudreault | Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration[END_REF] The pivotal clinical trials did not observe an evidence for retinal toxicity during the standard therapy with 0.5mg ranibizumab every four weeks in the treatment of wet AMD during an