MD Antonio Polito
email: apolito23@yahoo.it

MD Matteo Cereda

MD Federica Romanelli

MD Grazia Pertile

Macular Translocation with 360-Degree Retinotomy for Management of Retinal Pigment Epithelial Tear: Long-term Results

Keywords: Age related macular degeneration, retinal pigment epithelium tear, macula translocation

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Introduction

Tears of the retinal pigment epithelium (RPE) in the setting of neovascular age-related macular degeneration (AMD) have been described spontaneously [START_REF] Hoskin | Tears of detached retinal pigment epithelium[END_REF] or after various treatments, such as laser [START_REF] Gass | Retinal pigment epithelial rip during krypton red laser photocoagulation[END_REF] , photodynamic therapy [START_REF] Gelisken | Retinal pigment epithelial tear after photodynamic therapy for choroidal neovascularization[END_REF] , intravitreal triamcinolone [START_REF] Michels | Retinal pigment epithelium tears following verteporfin therapy combined with intravitreal triamcinolone[END_REF] and, more recently, intravitreal vascular endothelial factor (VEGF)-modulating therapy, including pegaptanib [START_REF] Singh | Retinal pigment epithelial tears after pegaptanib injection for exudative age-related macular degeneration[END_REF] , bevacizumab [START_REF] Chan | Retinal pigment epithelial tears after intravitreal bevacizumab injection for neovascular age-related macular degeneration[END_REF][START_REF] Chang | Tears of the retinal pigment epithelium: an old problem in a new era[END_REF][START_REF] Hannan | Retinal pigment epithelial tear following intravitreal bevacizumab for choroidal neovascular membrane due to age-related macular degeneration[END_REF][START_REF] Ronan | Retinal pigment epithelium tears after intravitreal injection of bevacizumab (avastin) for neovascular age-related macular degeneration[END_REF][START_REF] Weinberger | Retinal pigment epithelium tears after intravitreal bevacizumab in pigment epithelium detachment[END_REF][START_REF] Meyer | Acute retinal pigment epithelial tear following intravitreal bevacizumab (Avastin) injection for occult choroidal neovascularisation secondary to age related macular degeneration[END_REF][START_REF] Spandau | Retinal pigment epithelium tear after intravitreal bevacizumab for exudative age-related macular degeneration[END_REF] and ranibizumab [START_REF] Bakri | Retinal pigment epithelial tear after intravitreal ranibizumab[END_REF][START_REF] Carvounis | Retinal pigment epithelium tears following ranibizumab for exudative age-related macular degeneration[END_REF][START_REF] Maaijwee | Retinal pigment epithelium (RPE)choroid graft translocation in the treatment of an RPE tear: preliminary results[END_REF] . The most common lesion subtype associated with RPE tears is a fibrovascular pigment epithelium detachment (PED) or serous PED associated with choroidal neovascularization (CNV). Since most PED characteristics for spontaneous RPE tears, such as signs of underlying CNV (i.e., heme, exudate, notching, hot spot or irregular fluorescence) are similar to those of RPE tears following treatments, anti-VEGF therapy may accelerate the natural history of these lesions.

Patients often experience a sudden and severe loss of vision if the tear involves the fovea, with visual prognosis generally poor due to the scarring of fibrovascular tissue [START_REF] Hoskin | Tears of detached retinal pigment epithelium[END_REF] . Even when the fovea is not involved, and despite relatively intact visual acuity initially, long term functional prognosis is often 20/200 or less [START_REF] Chuang | Repair after tears of the retinal pigment epithelium[END_REF] . Moreover, these lesions are often bilateral, raising the issue of treating patients with one eye already affected and with high risk characteristics in the felloweye [START_REF] Chuang | Bilaterality of tears of the retinal pigment epithelium[END_REF][START_REF] Schoeppner | The risk of fellow eye visual loss with unilateral retinal pigment epithelial tears[END_REF] . Once the tear has occurred, no effective treatment exists, since current anti-VEGF therapies can not reconstitute a disrupted interface between photoreceptors and RPE.

The growing use of anti-VEGF therapy for neovascular AMD and the likelihood of an increase in RPE tears in a sub-population of eyes with high risk characteristics, together with a lack of treatment modalities, makes alternative treatments an important objective. Restoration of a healthy subretinal environment in eyes with RPE tears by relocating the fovea over apparently healthy RPE has the theoretical potential of improving visual function. This can be achieved by surgically removing the CNV and either rotating the macula to a new location with a 360° retinotomy or translocating an RPE-choroidal graft under the macula. Preliminary results of RPE-choroid graft translocation and single case reports on macular translocation with 360° retinotomy for RPE tears found vision restoration that was sustained for up to two years 15, 19-21 . This study presents the data of the first series of patients with an RPE tear with full macular translocation (FMT) with 360° retinotomy treated at our hospital and follow-up examinations of at least three years.

Materials and methods

Institutional review board approval was obtained for this study. The medical records of six eyes of six consecutive patients who had undergone FMT with 360° retinotomy for an RPE tear between March 2005 and June 2006 were retrospectively reviewed. One vitreoretinal surgeon (PG) performed all surgeries. All patients had a history of neovascular AMD and RPE tear in the study eye and advanced AMD with very limited visual acuity (BCVA < 20/400) in the fellow eye. Three patients had spontaneous RPE tear associated with fibrovascular PED and, in the other three, a tear had developed one month after intravitreal triamcinolone (IVT) (table 1).

FMT with 360° retinectomy was performed as initially described by Eckardt and collegues and later by Pertile and Claes [START_REF] Eckardt | Macular rotation with and without counterrotation of the globe in patients with age-related macular degeneration[END_REF][START_REF] Pertile | Macular translocation with 360 degree retinotomy for management of age-related macular degeneration with subfoveal choroidal neovascularization[END_REF] . Briefly, compensatory muscle surgery was performed to obtain a counter-rotation of the globe. Crystalline lens extraction with intraocular lens implantation into the capsular bag was performed on all patients by phacoemulsification through a corneoscleral incision. Complete pars plana vitrectomy was performed with posterior vitreous detachment and meticulous vitreous base shaving. Through a small posterior retinotomy, balanced salt solution was injected into the subretinal space with a 41-gauge subretinal cannula.

Detachment of the entire retina was accomplished after three or four injections followed by repeated fluid-air exchanges. A 360° retinectomy was performed as peripherally as possible with two specially-designed curved scissors (DORC International). The subfoveal lesion was removed.

The neurosensory retina was then rotated off the area of RPE and choriocapillaris loss; this was followed by an infusion of perfluorocarbon liquid. Both the small strip of retina remaining attached to the ora serrata and the excess of retina covering the pars plana in the superior nasal quadrant were removed with the vitreous cutter. Laser photocoagulation was applied to the retinectomy margins, followed by perfluorocarbon-to-silicone oil exchange. Silicone oil was removed in a second operation at 5.5 (2.1) (mean (SD)) months after surgery. Complete ophthalmic and visual acuity examinations with Snellen charts were performed periodically. Color fundus photography, fluorescein and ICG angiography and optical coherence tomography (OCT) were performed on all patients preoperatively, at 12, 24 and 36 months. During follow-up visits, OCTs were performed at all visits while angiographies were done only if recurrent CNV was suspected. The Snellen best-corrected distance visual acuity measurements were converted to the logarithm of the minimal angle of resolution (logMAR) units for analysis.

Results

The mean follow-up time was 39.2 months with a range of 36 to 50 months. All six patients presented the distinctive appearance of an RPE tear on biomicroscopy and fluorescein angiography in the treated eye (Figures A,B) and a disciform scar in the fellow eye. In all but one eye, the tears involved the fovea; the fovea was located above the folded RPE, clearly visible on OCT (Figure C). In patient 2 a large area denuded of RPE was located just inferonasal to the macular region, where a fibrovascular pigment epithelium detachment was present. In patients 3 and 5, a large subretinal hemorrhage covering more than 50% of the area of the entire lesion was also present. The subretinal hemorrhage obscured the area of folded RPE, but allowed clear visualization of the bare choroid and the corresponding window defect adjacent to the fovea on fluorescein angiography. All patients had visual disturbances within 3 months of surgery, except for patient 2, who complained of decreased vision after 1 year. Patient 2 had also been previously treated with IVT, photodynamic therapy and IVT at 6-month intervals between treatments at another clinic.

Best corrected visual acuity at baseline ranged from 20/2000 to 20/80 (mean 20/200). Baseline characteristics of the patients and the RPE tears in the six study eyes are summarized in Table 1. At 3, 6 and 12 months after surgery, best-corrected visual acuity had improved to a mean of 20/125, 20/80 and 20/63, with a mean increase of 6 lines of logMAR visual acuity at one year. At 24 and 36 months, mean visual acuity had improved to 5.57± 5.54 and 6.67± 4.76 lines over baseline, respectively, The improvement was maintained at the final visit (Table 2). All patients had at least 3 lines of improvement in visual acuity.

Fundus examination showed a dry macula and a pigment epithelial defect inferior to the fovea extending beyond the inferior temporal arcade at 1 month in all treated eyes. Fluorescein angiography at 1 year revealed a transmission defect inferior to the macula corresponding to the area of RPE defect caused by the RPE tear and surgical removal of choroidal neovascularization (Figures D,E). The extent of retinal rotation was sufficient to relocate the center of the fovea away from the atrophic zone in all but one eye (patient 2), which presented RPE atrophy extending toward the new fovea. This finding was also confirmed by OCT at one year (Figure F). OCT revealed a restoration of retinal morphology and foveal contour in all but one eye (patient 2), which showed a significant reduction in neurosensory retinal thickness with absence of outer retinal layers. After three years, a slight progression of RPE atrophy not extending to the new fovea could be observed ( Figures G,H

Discussion

RPE tears are frequently associated with severe visual loss due to their poor natural history and progressive subretinal scarring [START_REF] Chuang | Repair after tears of the retinal pigment epithelium[END_REF] . Moreover, patients affected by this lesion in one eye have a high risk of a similar event occurring in the fellow eye [START_REF] Chuang | Bilaterality of tears of the retinal pigment epithelium[END_REF][START_REF] Schoeppner | The risk of fellow eye visual loss with unilateral retinal pigment epithelial tears[END_REF] . While effective in improving vision in a large number of patients with neovascular AMD, anti-VEGF therapy does not improve vision in patients with RPE tears due to the irreversible loss of the integrity of the photoreceptor -RPE complex. In addition, this therapy appears to precipitate the occurrence of RPE tears in predisposed eyes [START_REF] Chang | Tears of the retinal pigment epithelium: an old problem in a new era[END_REF] . Although it has been noted that RPE tears are an infrequent complication of anti-VEGF therapy, an increasing number of reports have described such events after intravitreal injection of triamcinolone, pegaptanib, bevacizumab and ranibizumab . Unfortunately, no therapy has been shown beneficial once an RPE tear has occurred. At present, restoration of the subretinal anatomy with potential improvement of photoreceptor function can only be achieved by surgery. In fact, only through the surgical removal of the neovascular lesion can the fovea overlying the diseased macula be relocated into contact with an area of healthy-appearing RPE, either by macular translocation or RPE transplantation. This approach has the theoretical advantage of re-establishing the interaction between the RPE and photoreceptors which is fundamental to sight. In this study, we investigated the long term results in a consecutive series of six patients who underwent FMT for RPE tears in the study eye and suffered from severe visual loss due to advanced AMD in the fellow-eye. The extremely encouraging results of 5 to 16 logMAR lines of vision improvement at 1 year (mean 6 lines) were maintained at three years, when mean visual acuity was 20/50. Although in 2 eyes (patient 3 and 5) visual improvement may have also been influenced by removal of a large submacular hemorrhage, preoperative imaging clearly showed foveal involvement in the RPE tear implying a similar risk for further visual loss without FMT. Moreover, it is unlikely that the degree of visual improvement achieved in these patients could have been obtained by submacular hemorrhage displacement alone.

The outcomes of this series compare favourably with previous studies on FMT [START_REF] Pertile | Macular translocation with 360 degree retinotomy for management of age-related macular degeneration with subfoveal choroidal neovascularization[END_REF][START_REF] Mruthyunjaya | Change in visual function after macular translocation with 360 degrees retinectomy for neovascular age-related macular degeneration[END_REF][START_REF] Aisenbrey | Long-term follow-up of macular translocation with 360 degrees retinotomy for exudative age-related macular degeneration[END_REF] . This is probably due to timely surgical intervention with respect to the onset of RPE tear and vision loss -no more than 3 months in five of the six eyes, which may account for minimal retinal damage to the neurosensory retina and the increased possibilty of photoreceptor rescue and recovery of visual function. The only patient with visual symptoms of approximately one year duration (patient 2) was alone in having unchanged vision throughout the first two years and lower final visual acuity. A large atrophic RPE area extending toward the prospective fovea was also present in this patient. A recent paper on long term follow up of FMT has shown how progressive atrophy of the RPE presumably related to a more advanced stage of disease prior to surgery may significantly limit the beneficial effect of the treatment. Therefore early treatment seems a key factor in possible visual recovery. In addition, a good final anatomic result was achieved in all eyes, with few intraoperative and postoperative complications and with no incidence of proliferative vitreoretinopathy. Complications were successfully resolved in all cases.

Because of a limited inferior retinal detachment, patient 1 required an additional operation shortly after the first; patient 6 developed a choroidal haemorrhage during vitrectomy and FMT was completed a few weeks later, when the haemorrhage had been completely reabsorbed; patient 4 underwent laser photocoagulation followed by intravitreal bevacizumab for recurrent CNV 15 months after FMT. These outcomes support the observation that a refined surgical technique aimed at minimizing intraoperative and postoperative complications as well as the correct management of such events when they do occur are additional crucial factors for the achievment of improved visual acuity.

At present only three cases of RPE tears treated with FMT have been reported in the literature. In 2001 Meyer and Toth, in a report focusing on the pathomechanisms of RPE tears described a patient with RPE tear whose vision improved from 20/70 to 20/60 following FMT surgery, which was maintained at 15 months [START_REF] Meyer | Retinal pigment epithelial tear with vitreomacular attachment: a novel pathogenic feature[END_REF] . In 2004 Gelisken et al reported a case of RPE tear, with a 2-month history of decreased vision, treated with FMT; vision improved from 20/200 to 20/50 at 22 months with no PVR or recurrence of CNV [START_REF] Gelisken | Macular translocation with 360 degrees retinotomy for retinal pigment epithelial tear[END_REF] . In 2007 Gibran et al described a case of RPE tear following 2 bevacizumab injections, with vision decreasing from 20/40 to 20/320, no improvement was noted folowing a third bevacizumab injection and the eye was then treated with FMT surgery -final vision acuity was 20/80 at 7 months [START_REF] Gibran | Histological findings of a choroidal neovascular membrane removed at the time of macular translocation in a patient previously treated with intravitreal bevacizumab treatment (Avastin)[END_REF] . These three cases all showed visual improvement consistent with our findings. More recently, Maaijwee et al presented preliminary data on six patients with an RPE tear treated with RPE-choroid graft translocation [START_REF] Maaijwee | Retinal pigment epithelium (RPE)choroid graft translocation in the treatment of an RPE tear: preliminary results[END_REF] .

At final examination, between 6 months and 2 years post-operative, mean visual acuity improved from 20/160 to 20/80. The authors preferred this technique to FMT because it was faster and less complex. It also offered potentially fewer postoperative complications without the need for counter-rotation of the globe. However, with submacular insertion of an RPE-choroid patch graft, long-term gain in visual acuity has been achieved in a limited number of cases only and, when it does occur, improvement is minimal in the majority of patients, whereas many patients suffer significant vision loss. Our preliminary experience with this surgery in cases of RPE tears confirm such modest outcomes. Nevertheless, given the small number of patients and differences in surgical technique and surgeon experience, direct comparisons between these two treatments can not be made.

In conclusion, our findings show that FTM may represent an important therapeutic option to improve vision in patients with RPE tears of recent onset and a known natural history of visual deterioration. This study has several limitations, including its retrospective nature, the absence of a control group and the small number of patients. In particular, although we attempted to include consecutive patients during a definite time period, otherwise eligible patients may not have been selected because of lack of preoperative imaging. However, the long follow-up period enhances the clinical relevance of our encouraging results. Although this is a complex surgical procedure with potentially serious complications, the lack of response to anti-VEGF therapy, the absence of alternative treatments, the advanced disease of the fellow eye implying a similar risk for the affected eye, the continuous refinement of the surgical technique and the potential to acheive 20/80 or better visual acuity with early intervention, suggest that this treatment modality may have a role for the management of RPE tears in neovascular AMD. The Corresponding Author declares that there are not competing interests

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

  ) and preservation of foveal photoreceptor integrity could be confirmed by OCT in all eyes, with the exception of patient 2 (Figure I). Patient 1 presented a mild postoperative inferior retinal detachment, successfully reattached with one additional surgery with silicon oil tamponade 6 days after FMT. Patient 4 complained of diplopia two months after surgery, which spontaneously resolved at 3 months. Patient 6 developed a moderate choroidal hemorrhage during the cleaning of the vitreous base; the operation was suspended without performing the retinal separation and continued 17 days later, after the hemorrhage had been reabsorbed. Recurrent CNV occurred in patient 4 fifteen months after surgery, with vessels growing along the superonasal margin of the postoperative atrophic zone, approximately 1 disc diameter from the foveal center. In this patient CNV was successfully treated by photocoagulation of the feeder vessel and intravitreal injection of bevacizumab.

Figure. A ,

 A Figure. A, Preoperative colour photograph showing a retinal pigment epithelium (RPE) tear in a patient with neovascular age-related macular degeneration (patient 6, table 2). A large crescentic area of bare choroid is present temporal to the fovea with adjacent area of relative hyperpigmentation nasally, corresponding to retracted RPE. B, Preoperative fluorescein angiogram (FA) showing sharply demarcated transmission defect temporal to the fovea, consistent with the bed of the RPE tear and subfoveal choroidal neovascularization (CNV) within the area of retracted RPE. C, Preoperative OCT revealing irregularly elevated and folded hyperreflective RPE layer and break point temporal to the fovea (arrow). D, Fundus autofluorescence (FAF) at 12 months after surgery, with a visual acuity of 20/25. The FAF defect inferior to the macula corresponds to the area of RPE defect caused by the tear and surgical removal of CNV. This area appears as a hyperfluorescent window defect on FA (E). F, OCT at 12 months showing a normal foveal anatomy over a healthy appearing RPE layer. G and H, FAF and FA at 36 months after surgery, visual acuity maintained at 20/25. Note the slighlty increased RPE defect, but still not involving the new fovea. I, Spectral Domain OCT showing preservation of macular architecture, with clear visualization of the intraretinal microstructure of the new fovea and in particular the photoreceptor inner and outer segment junction (arrow).

Table 1 .

 1 Clinical characteristics of the six patients with a retinal pigment epithelium (RPE) tear treated with a full macular translocation with 360° retinotomy

Table 2 .

 2 Best corrected visual acuity (Snellen and LogMAR) change after surgery

	Patient	Baseline	1 month	3 months 6 months 12 months	24 months	36 months
	No.
	1	20/2000	20/1250	20/100	20/40	20/50	20/50	20/50
		2.0	1.7	0.7	0.3	0.4	0.4	0.4
	2	20/400	20/400	20/400	20/400	20/400	20/400	20/125
		1.3	1.3	1.3	1.3	1.3	1.3	0.8
	3	20/125	20/63	20/50	20/50	20/32	20/40	20/40
		0.8	0.5	0.4	0.4	0.2	0.3	0.3
	4	20/200	20/400	20/200	20/200	20/80	20/80	20/80
		1.0	1.3	1.0	1.0	0.6	0.6	0.6
	5	20/100	20/125	20/125	20/40	20/32	20/40	20/32
		0.7	0.8	0.8	0.3	0.2	0.3	0.2
	6	20/80
		0.6


MD Florian Gekeler
email: gekeler@uni-tuebingen.de

MD Andreas Kopp

MD Helmut Sachs

MD Dorothea Besch

Udo Greppmaier

MD Eberhart Zrenner

MD Karl Ulrich Bartz-Schmidt

MD Peter Szurman

Visualization of active subretinal implants with external connections by high-resolution computed tomography

Keywords: retinal implant, prosthesis, computed tomography, subretinal F. Gekeler

2) RIAG covered the direct costs of the study;

(3) HS, EZ and FG are shareholders of RIAG; none of them receives payment or is employed by RIAG.

Introduction

Subretinal implants intend to restore visual function in diseases such as retinitis pigmentosa (RP) by replacing degenerated photoreceptors with microphotodiode arrays (MPDAs) [START_REF] Chow | The subretinal microphotodiode array retinal prosthesis [letter; comment[END_REF][START_REF] Zrenner | Will retinal implants restore vision[END_REF][START_REF] Zrenner | Can subretinal microphotodiodes successfully replace degenerated photoreceptors?[END_REF] . Since it has been shown that more energy is necessary than is available through microphotodiodes [START_REF] Stett | Electrical stimulation of degenerated retina of RCS rats by distally applied spatial voltage patterns[END_REF] active subretinal implants with external connections have been constructed 5 6 . In this first clinical trial, a trans-cutaneous access in the retro-auricular space provided important advantages: it allowed direct evaluation of electrode parameters such as impedance and charge transfer; and second, it provided maximum security and stability for stimulation and driving circuits of the subretinal device. Future implants, however, will be fully implantable with wireless transmission of signals and energy.

After implantation, ophthalmologic examination is limited to external aspects and to the ocular fundus where the implant can be directly seen up to the point of trans-choroidal passage.

However, the subdermal cable, the connection of the cable to the polyimide foil in the temporal fossa, and the passage of the foil around the lateral orbital rim through the orbit to the transchoroidal passage, all of these evade direct examination. Some groups applying epiretinal prostheses use similar extra-ocular routes, 7 8 while other groups prefer an all-intraocular approach [START_REF] Roessler | Implantation and explantation of a wireless epiretinal retina implant device: observations during the EPIRET3 prospective clinical trial[END_REF] .

In order to visualize an implant with extra-ocular parts in vivo, to evaluate it under strong eye movements, and to examine the tissue surrounding it, we have performed this exploratory study using high-resolution computed tomography (CT).

Patients, Material and Methods

Two patients were included, both were male and suffered from end-stage RP with light perception. They gave written informed consent; all procedures adhered to the Declaration of

Results

CT scans were obtainable in high quality with only minor adjustments to regular examination parameters for thin slice orbital exams. Virtually all parts of the implant were clearly visible: the subretinal part with the 4*4 array and the thicker portion resembling the MPDA, the golden connection lanes on the polyimide foil in the subretinal space following the curvature of the posterior eyeball, its bend at the choroidal/scleral passage, the fixation pads for episcleral suturing, the foil's path through the orbital cavity and around the lateral orbital rim, the connection pad to the silicone cable in the temporal fossa, the silicone cables way to the retroauricular space, and the metal fixation clamp on the skull. Fig. 4 shows 3D-reconstructions of P1 from different angles along a transversal plane around the implant. In 3D reconstructions relative dimensions of various implant parts were evaluated to assess if such measurements would yield meaningful results. The MPDA measured exactly 3 mm * 3 mm and was taken as reference in its largest presentation in CT images. The silicone cable measured 3.19 mm (3 mm in reality; always averages of various measurements), the connection pad 9.6 mm (12 mm in reality; however, this structure was curved in the patient which was not measurable), the wider polyimide foil at the orbital rim 2.7 mm (2.5 mm in reality), the subretinal polyimide foil 1.5 mm (1.5 mm in reality). in photographic gaze analysis, but were not described as disturbing [START_REF] Besch | Extraocular surgery for implantation of an active subretinal visual prosthesis with external connections: feasibility and outcome in seven patients[END_REF] .

Under ftp://134.2.124.64/home (login and password: 'BJO') a self-maneuverable 3D-animation can be downloaded and viewed using Quicktime® (Apple Inc., Cupertino, CA, USA).

Discussion

CT has proven feasible in two patients with subretinal implants yielding strikingly illustrative pictures. CT is cost-effective and can provide higher detail than any other technique including ultrasound sonography, which also demonstrates bad penetration depth in bone, and magnetic resonance imaging, which is not recommendable for such kind of implants with cable wiring due to possible danger to implants and surrounding tissue through high-frequency electro-magnetic fields. While radiation exposure of CT scans, especially if performed frequently for study purposes, must be mentioned, CT constitutes the only means to visualize the implant's parts outside of the eye in high resolution which in our case can be estimated to be around 0.1 mm. CT images are readily transferrable to construct a 3D image in external software. In these, meaningful measurements of dimensions of the implant could be performed.

CT pictures of such high resolution can show all technical aspects of the implant and give valuable indications for research purposes and in case of technical problems. Images also deliver data on the tissue reaction (biocompatibility) and can visualize the behaviour of the implant under eye movements. And, last but not least, such three dimensional images and animations are excellent for illustrations and instructional purposes.

Epiretinal as well as subretinal implants currently mostly utilize extra-ocular connections for energy and signal conduction. Since they are still implanted under clinical trial conditions adequate monitoring tools are mandatory to study the integrity of the device (biostability) and the body's reaction to it (biocompatibility). We believe that high-resolution CT scans can contribute to the success of these implants by delivering adequate imaging material. (b) A clamp was built from surgical steel for fixation of the implant on the scull bone in the retroauricular region. The clamp consisted of a base plate which was screwed into the scull bone and a cover plate with a recess to jam the silicone cable onto the base plate (scale is in mm).

Figure 2

Original transversal CT images of P1. a. In this section the foil with gold connection lanes around the lateral orbital rim, within the orbit and its course on the eyeball with the scleral fixation can be seen. In the region at the lateral orbital rim there is moderate soft tissue proliferation surrounding the implant in comparison to the contra-lateral side. All other orbital structures in this and sections b-d of this figure, including sclera, optic nerve, and eye muscles are normal. b. In this section the course of the polyimide foil with the gold connection lanes can be followed from the orbital rim; on the posterior pole the implant's tip in the subretinal space is visible. c. This section shows the full extent of the subretinally implanted microphotodiode-array with a size of approximately 3 mm. The scans were taken 21 months after implantation; the external parts of the implant and the metal clamp had been removed 12 months after implantation when granulation tissue had occurred at the skin perforation site and the implant was not used any more due to regulation by the ethics committee. -All parts of the implant are clearly visible: 1. the subretinal tip with an MPDA and the direct stimulation electrodes (coloured blue; colors do not represent differences in Hounsfield units, but rather were applied later to a 3D raw image serve better visualization), 2. the fixation pads for episcleral fixation of the polyimide foil (red), 3. the orbital part of the polyimide foil, 4. the connection of the foil to the silicone cable in the temporal fossa, 5. the silicone cable leading subperiostally to the retro-auricular space (green). The scans were taken 2 months after implantation; the implant was complete with the clamp in the retro-auricular space and its external parts. -All parts of the implant are also clearly visible as in fig. 1 and have been coloured identically; in addition, the metal clamp is coloured turquoise.

Figure 6

Orbital details of CT scans in 3 D reconstructions under different directions of gaze.

Of the 9 directions of gaze scanned only those where implant movements are extreme are shown, i.e. in direction or away from the direction of the orbital entry point. The implant's movements can be detected by comparing the position of the blue subretinal tip and the episcleral fixation pads of the polyimide foil (red).

Quicktime® animation (downloadable)

Similar images as in fig. 1 were used to compose this self-maneuverable animation which additionally allows cranial, caudal, and oblique views. Please go to ftp://134.2.124.64/home (login and password: 'BJO'; Quicktime® is obligatory to maneuver the images).

  P1; 44 years old) was implanted in October 2005 in his right eye. Although the study period and insurance covering was granted for 4 weeks by the local ethics committee the patient refused explantation; he still carries the implant. The device's extra-corporeal parts in the retroauricular area were removed and the skin closed after 12 months. P2 (42 years old) was implanted in October 2007. Because of beginning implant failure the extraocular parts were removed three months later (the study period was granted for 4 months at this point) and the intra-ocular parts 10 months post-operatively on the patient's decision. The implant consisted of 4 connected parts (details are given in fig.1). The subretinally placed tip comprised two different entities: a MPDA with 1,500 microphotodiodes for light-driven stimulation and a 4*4 electrode array for direct electrical stimulation. A SOMATOM Definition® (Siemens AG, Forchheim, Germany) CT scanner was used, collimation was 0.5 mm at 120 kV and 100 mA. DICOM (Digital Imaging and Communications in Medicine) images of P1 were segmented using Amira® (Visage Imaging GmbH, Berlin, Germany), exported as VRML (virtual reality modeling language)-data and rendered in Maxon Cinema 4D® (Maxon Computer GmbH, Friedrichsdorf, Germany). Images of P2 were segmented and rendered by the CT scanner's Leonardo® workstation software (Siemens AG, Erlangen, Germany) to generate 3 D images. Final images and colors were obtained using Photoshop® (Adobe Systems Incorporated, San Jose, CA, USA). Scans were taken in primary position (patient looking straight) with eyes open and eyes closed, and in 8 cardinal directions of gaze: up, down, left, right, upper right, upper left, lower right, lower left.

Fig. 2

 2 Fig.2shows 4 transversal sections of P1 with moderate soft tissue reaction to the orbital parts of the implant near the lateral orbital rim; all other structures within the orbital cavity, including sclera, optic nerve and eye muscles seem unaffected.

Fig. 3

 3 Fig. 3 shows the steps to segment an original 2 D image to obtain a 3 D image as shown in figs. 4-6.

Fig. 5

 5 Fig. 5 shows similar reconstructions of P2 who still carried the entire implant showing also the metal clamp and the extra-corporeal part.

Fig. 6

 6 Fig. 6 shows 4 views of P1 at three different directions of gaze: straight, and upper right and lower left causing maximum deflections in the orbit. Eye movements were minimally restricted

FFigure 1

 1 Figure 1Photographs of (a) the subretinal implant and (b) the fixation clamp.

F

  . Gekeler et al.: CT visualization of subretinal implants d. In this section the connection pad in the temporal fossa produces the typical radiating artifacts from metal.

Figure 3

 3 Figure 3Steps for segmentation of original images to obtain 3D reconstructions. a. This screenshot shows one original image on the left side which was chosen from the sections on the right side. b. This screenshot shows the segmentation tool of the software; the segmented parts consisting of bone and implant are shown in red. c. This screenshot shows one raw version of a 3 D reconstruction from segmented images.

Figure 4 3

 4 Figure 4 3 D reconstructions of CT scans of patient 1.

Figure 5 3

 5 Figure 5 3 D reconstructions of CT scans of patient 2 with a subretinal implant.

F. Gekeler et al.: CT visualization of subretinal implants


Mona Khandwala

Penmetsa Bhanu Rakesh

MSc Bsc

MBBS J B Sarju Dey

FRCPath Schofield

A Carole

FRCS, FRCOphth Jones

PhD Adrian Podoleanu

Tunbridge Wells

Nhs Trust

M Khandwala
email: mona.khandwala@gmail.com

Imaging of periocular basal cell carcinoma (BCC) using en-face optical coherence tomography (OCT)-A Pilot Study

Keywords: Carcinoma, Basal Cell; Optical Coherence Tomography, BCC, OCT Word count: 2628

Aim

To use en-face optical coherence tomography (OCT) imaging to identify features of tumour tissue and their correlation with histopathology, and to assess the effect of different wavelengths and resolutions of OCT in identifying tumour boundaries and features.

Conclusion

En-face OCT imaging has the potential to identify tumour tissue from healthy tissue. It also showed correlation with corresponding histopathology findings. Non contact OCT imaging of the skin is a non invasive and convenient method and can be useful for demarcating BCCs on the face and eyelids. Future larger studies on in vivo BCCs using en face ultra high resolution OCT should provide information on sub typing of BCCs.

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 (http://bjo.bmj.com/ifora/ licence.pdf).

Methods

Excision specimens of consecutive biopsy-proven periocular BCCs (n=8) were assessed by OCT performing in vitro cross section scans and en-face scans of the tissues. Images were collected from three different machines; systems 1 & 2 had a wavelength of 1300 nm and system 3 had a wavelength of 840 nm. System 2 used high numerical aperture (NA) interface optics which determines higher magnification and hence allows higher transversal resolution. All 8 specimens subsequently underwent routine histopathology examination.

Results

Three common features of tumour tissue were observed in all three systems: (i) lobular pattern of abnormal architecture, (ii) dilated blood vessels and (iii) high reflective margins. We compared the three systems based on their ability to pick up the three above mentioned tumour features. In this respect, system 2 had the highest capability in picking up feature (i), followed by systems 1 and 3. In terms of feature (ii), similar results were obtained with all three systems. System 3 was unable to pick up feature (iii), whereas Systems 1 and 2 performed equally.

INTRODUCTION

Basal cell carcinoma (BCC) is the most frequently occurring periocular malignancy, with a rapidly increasing incidence worldwide, both because of increased sun exposure and longevity. [START_REF] Allali | Basal cell carcinoma of the eyelids[END_REF] Surgical excision with predetermined margins 2 , or Mohs' surgery where available [START_REF] Mohs | Micrographic Surgery for the Microscopically controlled excision of eyelid cancers[END_REF][START_REF] Margo | Basal cell carcinoma of the eyelid and periocular skin[END_REF][START_REF] Cook | Treatment options and future prospects for the management of eyelid malignancies -An evidence-based update[END_REF] , is recommended to ensure complete tumour removal.

Several non invasive diagnostic methods have been reported so far but none have yet proved to be practical. High frequency ultrasound, terahertz imaging and magnetic resonance all offer limited resolution. [START_REF] Woodward | Terahertz pulse imaging of ex vivo basal cell carcinoma[END_REF][START_REF] Buchwald | Optical coherence tomography versus ultrasound microscopy of conjunctival and eyelid lesions[END_REF][START_REF] Gufler | High-resolution MRI of basal cell carcinomas of the face using a microscopy coil[END_REF] Reflectance confocal microscopy produces high quality images but it has the disadvantage of requiring contact with the tissue, and having a limited field and depth penetration. Ultrasound provides better penetration, but has a lower resolution when compared to confocal microscopy. [START_REF] Brezinski | Assessing atherosclerotic plaque morphology: comparison of optical coherence tomography and high resolution intravascular ultra sound[END_REF] Optical coherence tomography (OCT) is a novel imaging technology 10 used to determine the structure of translucent objects. It combines a high axial resolution with good tissue penetration. Whilst well established for retinal imaging [START_REF] Podoleanu | Combinations of techniques in imaging the retina with high resolution[END_REF] , the equipment currently used in ophthalmology clinics does not provide reliable skin images. OCT has been successfully used for high resolution imaging of structures in other parts of the body [START_REF] Bibas | 3-D optical coherence tomography of the laryngeal mucosa[END_REF][START_REF] Wang | Polypropylene Glycol A Contrasting Agent for Optical Coherence Tomography to Image Gastrointestinal tissues[END_REF] and has been successfully employed to identify tumour features and abnormal skin architecture. [START_REF] Olmedo | Optical coherence tomography for the characterization of basal cell carcinoma in vivo: A pilot study[END_REF] In this paper we assess the use of en-face OCT, a time domain variant of the OCT technology to identify and delineate periocular BCCs.

METHOD Clinical Study

A pilot study to assess the capability of OCT to delineate tumour from normal tissue was performed. Ethical approval for the study was obtained from both the Ethics Committee of the Faculty of Science Technology and Medical Studies, University of Kent and the West Kent Ethics Committee. The research described adhered to the tenets of the Declaration of Helsinki.

In this study, excision biopsies were performed on periocular BCCs by members of the Ophthalmology Department at Maidstone and Tunbridge Wells NHS Trust. Patients attending the Ophthalmology department at Maidstone Hospital were recruited to the study after informed consent was obtained. All 8 patients recruited had a biopsy-proven BCC within the periorbital region. After routine surgical excision using predetermined tumour margins the tissue was marked with sutures at superior, medial and lateral margins using 5/0 silk (Ethicon). Within 3 hours of excision, the samples were transported in formaldehyde to the Applied Optics Group Lab within the University of Kent for OCT imaging. The technology required for OCT imaging of skin is at a much earlier stage of development than that for retinal imaging. All samples were scanned by three different OCT machines. These are experimental prototypes, thereby allowing in vitro examination only. The origin of the samples and patient details were single masked from the group performing the OCT scans.

For scanning, the samples were fixed onto pieces of polystyrene board to align the longest ends of the samples along the horizontal direction. Images were obtained in vitro using each of the three OCT systems. Multiple en-face scans were obtained at steps of 25 μm in depth and horizontal cross section scans were obtained by moving the sample in the en-face plane (mostly along the vertical direction). On each of the three OCT machines, the scanning process took between 45 to 60 minutes, including the set up time. All the tissue samples were kept hydrated during the scanning process using drops of formaldehyde. This part of the protocol was agreed with the histopathologist prior to starting the study, ensuring preservation of the tissue for accurate histopathological analysis. On completion of the OCT scans, the tissue samples were stored in formaldehyde and processed for routine histological analysis, undertaken by a single histopathologist. After fixing the specimen, it was transversely sectioned "bread sliced" into several pieces which were processed and embedded in paraffin wax. 2-3 micrometer slices were obtained and stained with haematoxylin and eosin. The histopathological sections were therefore orientated in the axis of the OCT cross section scan. The pathologist was masked from the findings of the OCT scans, until routine histology reporting was done. Once histopathological analysis was completed on all 8 specimens, the OCT team and histopathologist examined all histopathology sections, to identify a section that correlated with the OCT image for each sample. Where it was not possible to exactly match the histopathological section to the OCT image, a section was chosen that displayed corresponding morphological features.

OCT instrumentation

OCT is based on a Michelson interferometer [START_REF] Fercher | Optical coherence tomographyprinciples and applications[END_REF] , where the light emitted by a source is split into an object beam and a reference beam using a beam splitter. The reference beam is reflected back from a reference mirror. Both beams are combined and the interference pattern read by an interferometer. Depth scanning is achieved by moving the reference mirror, lateral scanning is performed by moving the scan beam laterally, using a dual scanning head (equipped with two galvanometer scanners) and a lens. Enface OCT as used here generates lateral reflectivity profiles, called T-scans, obtained by scanning the beam horizontally over the target. [START_REF] Podoleanu | Transversal and longitudinal images from the retina of the living eye using low coherence reflectometry[END_REF]

DISCUSSION

Previous attempts to produce skin images using OCT have been reported [START_REF] Olmedo | Optical coherence tomography for the characterization of basal cell carcinoma in vivo: A pilot study[END_REF][START_REF] Bechara | Histomorphologic correlation with routine histology and optical coherence tomography[END_REF] but this technology is still under review. There are a lack of systematic studies which prove the efficacy of OCT as a tool for the diagnosis and delineation of BCC and all the studies undertaken to date have examined B-scan images only. Live en-face images provide a transverse view at a desired depth. This allows identification of all four margins at any particular depth of tissue. Combining the interpretation of B-scan and C-scan OCT images, the tumour margins can be better delineated in all three dimensions, as achieved by Mohs' surgery. In the future, this could be further enhanced using computer imaging to produce a 3D reconstruction of the tumour tissue. To the best of our knowledge, this is the first paper to look at en face OCT imaging in examining BCCs.

In this study, we observed the tumour margins as signal intense lines separating the tumour from the surrounding tissue and also dividing the tissue into different lobules.

Tumour tissue from the deeper layers of the skin appeared as oval and round, low-signal areas with no clear arrangement. The epidermis was clearly distinguishable from the dermis in samples where skin surface was even and the border was seen as a low reflective band. OCT images showed areas of high reflectivity corresponding to tumour lobules on histology. In addition, scattered high reflective areas surrounded by low reflective areas were observed within the abnormal tissue. Similar observations have been made in a previous study. Bechara et al [START_REF] Bechara | Histomorphologic correlation with routine histology and optical coherence tomography[END_REF] reported similar morphologic structures and formations both in histology and OCT, in their series of 6 patients with BCC and naevi. In their study the OCT images were taken in vivo, prior to lesion excision.

Although in vivo imaging has obvious clinical benefits, our study was limited to in vitro imaging due to the prototype setup of the machines available. Strasswimmer et al [START_REF] Strasswimmer | Polarization-sensitive optical coherence tomography of invasive basal cell carcinoma[END_REF] devised a pilot study using polarization sensitive OCT to identify features of BCC. Prior to surgical excision, they scanned different portions of the tumour. A loss of birefringence was noted in the tumour itself which was more marked in the infiltrative type of BCC. They were unable to correlate exact features of OCT and histology to determine precise lateral tumour extension.

It is recognised that water content and temperature of the body tissue can affect image quality. [START_REF] Troy | Optical properties of human skin in the NIR wavelength range of 1000-2200 nm[END_REF] Normal skin temperature is 37 degrees Celsius and it contains 60-70% water.

These constants are affected in excised tissue and can modify the optical properties of the skin. Since the samples used in our study were examined in vitro the temperature difference from normal body conditions might have played a significant part in the modification of tissue optical parameters. Schmitt et al [START_REF] Schmitt | Multilayer model for photon diffusion in skin[END_REF] have shown that differences exist in the optical properties when measurements are taken at different temperatures.

En-face (C) scans and cross sections (B) were performed on all 8 samples, with all three OCT systems; this provided the advantage of being able to correlate information at a particular depth in the C-scans with that along depth in B-scans, guiding us in delineating the margins of the tumour in all directions in 3D volume. B-scan OCT images may lead to missed features across the lateral aspect of the tumour.

There are no studies currently available which have reported en-face scans on basal cell carcinoma. In traditional B-scan OCT images, margins appeared as high reflective bands separating the tissue into segments. No such findings were demonstrated in previous studies on BCC. [START_REF] Strasswimmer | Polarization-sensitive optical coherence tomography of invasive basal cell carcinoma[END_REF] In this study, altered skin architecture was the most common finding in the majority of the samples and it had varied presentations.

Given the low contrast of OCT images, the way forward is to combine imaging modalities. Some encouraging results have been reported using fluorescence or polarisation. Thus simultaneous or sequential imaging in different regimes where the hardware allows accurate superposition of images could be a way forward.

In terms of ideal parameters, the wavelength of 1300 nm seems the most appropriate.

Radiation of longer wavelength may have better penetration, however the water absorption peaks up and the transversal resolution deteriorates. Radiation of shorter wavelength encounters more scattering and perhaps this explains our results here with the third machine at 840 nm being less effective. As regards the optimum regime of operation, time domain (TD) versus spectral domain (SD), it is largely accepted that SD-OCT offers better sensitivity than TD-OCT. However, SD-OCT acquires images under a fixed focus and as a consequence it is incompatible with higher magnification which requires focus change in synchronism with depth exploration. Higher magnification is also required for improved transversal resolution. Therefore, the enface OCT (a TD-OCT method), which allows focus change, seems better suited to investigations which require higher magnification, despite its lower sensitivity in comparison to SD-OCT. Lastly, considering the issue of image size versus transversal resolution, perhaps different probes with different magnifications may overcome the optics limitation which prevents good transversal resolution when under low magnification.

There were two main limitations to our study. The first was the small sample size. It was designed as a pilot study to demonstrate proof of concept, with a view to a larger study in the future depending on the results. The technical and logistical difficulties of transporting the samples to a different site for OCT scanning, then back to the lab for histopathological analysis, necessitated a small sample size. The other limitation was the in vitro scanning. Ideally, the study would have looked at in vivo scanning, as this is where the clinical application of OCT lies, but we were limited by the equipment available to us. A further study is planned to compare the results of in vitro versus in vivo, once the technology is available.

In conclusion, en-face imaging has the potential to demarcate tumour tissue from healthy tissue. It has also showed correlation with corresponding histopathology, as demonstrated in the figures above. Non contact OCT imaging of the skin is a very convenient method and may be very useful for imaging BCCs on the face and eyelids.

However, larger studies of BCC in vivo are required for better identification of tumour margins and sub typing tumours.

LEGENDS

  Multiple T-scans acquired at 500 Hz, are recorded for sequential values of the vertical coordinate to form an en-face OCT image (a 2D map, termed as a C-scan, or constant depth image). The vertical scanning is obtained by driving the frame galvo-scanner in the dual scanning head to move the beam vertically at 2 Hz. By changing the position of the reference mirror, C-scan images at different depths can be obtained, and for sufficient dense sampling of the tissue, 3D volumes can be reconstructed. En-face OCT has the advantage that by simple re-routing of the signals driving the two scanners, the system can be switched from Cscan into B-scan regime. A B-scan is obtained by stopping the frame scanner and by moving the reference mirror to explore the object in depth. In this way, B-scans are built as 2D-assemblies of T-scans repeated at sequential depths. B-scans represent cross section slices in the tissue, they are orthogonal on C-scans and represent the traditional OCT view. However, the en-face OCT method provides the added advantage of producing images with the orientation of microscopy images (C scans). Features with en-face orientation allow easier identification of tumour margins when compared to B-158 scan OCT images. All three systems used here can be operated in either C-scan or B-159 scan regime and their details are specified

Fig 3

 3 Fig 3 shows lobular pattern of abnormal architecture, as well as high reflective bands.

Fig 1 :

 1 Fig 1: (A) Horizontal B-scan OCT image obtained with system 1, image depth 1.5 mm

Fig 2 :

 2 Fig 2: (A) C-scan OCT image of biopsied tissue, obtained with system 2. Large low

Fig 3 :

 3 Fig 3: (A) C-scan OCT image obtained with systems 1, showing high reflective margins

Fig 4 :

 4 Fig 4: (A) C-scan OCT image of biopsied tissue obtained with system 3. Dilated blood

Table 1 .

 1 in Table1. Specifications of the three OCT systems used in the study

	160
	Central wavelength	Optical bandwidth	Superluminescent diode (SLD) power	Lateral resolution	Axial resolution
	System 1 1300 nm	60 nm	<1mW	>15 μm	13.6 μm
	System 2 1300 nm	60 nm	<1mW	<5 μm	13.6 μm
	System 3 840 nm	50 nm	<1mW	>15 μm	8.8 μm
	161
	162
	RESULTS
	170
	171			Pick up rate of tumour features
	(i) Lobular pattern of abnormal	(ii) Dilated blood vessels	(iii) High reflective
	172 System 1	architecture 5/8 (62.5%)	1/8 (12.5%)		margins 3/8 (37.5%)
	System 2	6/8 (75%)	1/8 (12.5%)		3/8 (37.5%)
	173 System 3	2/8 (25%)	1/8 (12.5%)		0/8
	Fig 1 illustrates the appearance of normal skin in an OCT image in comparison with
	176

163

Three common features of tumour tissue were observed with all the three systems. 164 These features were: (i) a lobular pattern of abnormal architecture, (ii) dilated blood 165 vessels in the upper dermis and (iii) high reflective margins of tumour lobules. Table

2

166 shows detailed results for each system. System 2 showed the best performance in 167 picking up the abnormal architecture, followed by systems 1 and 3. There was no 168 difference between systems in exhibiting dilated blood vessels. However, system 3 169 could not identify high reflective margins in any samples, while systems 1 and 2 could. 174 histology with haematoxylin and eosin stain. All the scans showed a well defined 175 epidermis. However, the thickness of the epidermis varied across the samples.

Table 2 .

 2 Pick up rates of tumour features for each of the three OCT systems 8 The typical lobular architecture of BCCs was noted both on histology and on OCT images. Details are as shown in Fig 2.

Also, Bonner et al [START_REF] Bonner | Model of photon migration in turbid biological media[END_REF] reported that the absorption spectrum of water in the near infra red region depends on temperature.

Different wavelengths for imaging the BCC samples were selected (1300 nm and 840 nm) taking into consideration the fact that the absorption coefficient for different layers of the skin is different. The second OCT system with high NA interface optics, which enables a high transverse resolution of better than 5 μm and axial resolution of 13.6 μm, was able to image tumours in greater detail. However, higher magnification and better transverse resolution result in a smaller field of view. In our study, the highest tumour detection rate was obtained with system 2 (high NA) followed by system 1 both operating at 1300 nm. System 3, which had a wavelength of 840 nm, did not show significant findings. This may be due to the reduced penetration depth at 840 nm as well as to a variation of contrast with wavelength.

Previous studies with OCT imaging of BCC showed features similar to those found in our study. [START_REF] Podoleanu | Transversal and longitudinal images from the retina of the living eye using low coherence reflectometry[END_REF][START_REF] Strasswimmer | Polarization-sensitive optical coherence tomography of invasive basal cell carcinoma[END_REF] Our study examined BCCs exclusively from the face and eyelids, used two different wavelengths and also performed en-face scans. Boundaries demarcating the tumour from normal tissue, as well as dividing the tumours into different segments, were clearly visible in the en-face images at 1300 nm wavelength. The disappearance of this feature at 840 nm could indicate a reflectivity and absorption profile for the tumour tissue. Altered architecture of the skin was seen very prominently in images from system 2 which had a transverse resolution comparable to what can be achieved in confocal microscopy.

COMPETING INTERESTS

Adrian Podoleanu: acted as consultant for, and has patents with, Ophthalmic Technology Inc, Canada and University of Kent.

FUNDING

No financial support given for the research described in this paper.


INSULIN RESISTANCE AND DIABETIC MACULAR EDEMA IN TYPE 2 DIABETES MELLITUS

Objective: To evaluate the association between insulin resistance and diabetic macular edema in type 2 diabetes.

Methods: Cross-sectional study. Type 2 diabetes patients who agreed to undergo blood sample extraction were enrolled. Patients who had received treatment for macular edema within the past 3 months and those with other retinal diseases were excluded. The following data were recorded: age, sex, time of diabetes evolution, HbA1C, ophthalmologic and systemic treatment, and lens status. Optical coherence tomography (OCT) was performed to determine the morphologic patterns of macular edema. Insulin resistance was established on a McAuley index of <6.3 and fasting insulin levels of >16 mU/L.

Results: 177 eyes from 90 patients were included; 27.1% of eyes were from insulin-resistant patients. There were no differences in age, time of evolution, sex, HbA1C level, or lens status between insulin-resistant and nonresistant patients. Insulin-resistant patients were more likely to have exogenous insulin therapy (P<0.05; OR=3.8). An association was found between diabetic cystoid macular edema and resistance (Fisher exact test P=0.007; OR=2.53, 95% CI: 1.52-4.2). There were no associations between insulin resistance and the other morphologic patterns of edema. Patients undergoing insulin therapy were found to have an association with a diffuse retinal thickening pattern on OCT (P=0.036; OR=1.4). However, no association was found between insulin therapy and the presence of cystoid macular edema.

Conclusions: The findings of this study indicate a relationship between insulin resistance and cystoid macular edema, which is unrelated to the use of insulin. Insulin treatment was associated with diffuse macular edema.

INTRODUCTION

Type 2 diabetes mellitus is the most common metabolic disease worldwide.

Whereas low plasma insulin levels are seen in type 1 diabetes mellitus, insulin resistance is a characteristic feature in type 2 patients. Macular edema is a major complication of diabetes and is the leading cause of blindness in patients aged 25 to 74 in developed countries.

The origin of diabetic macular edema resides in a series of local and systemic injuries to the retina, including vascular dysfunction, neural degeneration, and retinal inflammation. It remains unclear whether diabetic retinopathy is initiated by vascular dysfunction leading to neural degeneration and subsequent inflammation, or by dysregulation of the neural retina metabolism with subsequent inflammation and vascular damage [START_REF] Gardner | Novel potential mechanisms for diabetic macular edema: leveraging new investigational approaches[END_REF].

The relationship between diabetes mellitus and inflammation was first recognized more than 60 years ago [START_REF] Menkin | Diabetes and Inflammation[END_REF][START_REF] Ke | Inflammation, stress and Diabetes[END_REF], and recently an association between insulin resistance and systemic inflammation was reported [START_REF] Shoelson | Inflammation and insulin resistance[END_REF][START_REF] De Luca | Inflammation and insulin resistance[END_REF].

Abnormalities in glucose and lipid metabolism, obesity, and high blood pressure occur together commonly enough in the same individuals as to suggest that they are somehow interrelated. This cluster of metabolic abnormalities is attributable to insulin resistance; that is, a reduced sensitivity to the action of insulin in tissues. In the early stages of this condition there is a compensatory state of hyperinsulinemia, and later on, hyperglycemia. Insulin resistance is an inherently proinflammatory state that begins before the onset of overt hyperglycemia and is related in part to excess nutrients in adipose tissue, skeletal muscle, and the liver.

Inflammation induces a catabolic state that counteracts normal anabolic processes, probably as an adaptive mechanism to prevent excessive cellular nutrient uptake. Systemic inflammation increases with the onset of clinical diabetes and contributes to the development of complications, including nephropathy and retinopathy. In addition, high insulin levels have a vasoreactive effect that results in increased permeability of the retinal vessels [START_REF] Vassiliki | Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxiainducible factor-1 alpha and VEGF[END_REF][START_REF] Zapata | Blurred Vision and increased macular volume in the introduction of insulin therapy: a prospective study[END_REF].

Insulin resistance has been investigated and correlated with clinical risk factors, mainly in nondiabetic patients. Direct methods (the reference standard) can be used to measure insulin resistance, but they are complex to perform and expensive. Indirect measures of insulin resistance, such as fasting insulinemia [START_REF] Fontbonne | Hypertriglyceridemia as a risk factor of coronary heart disease mortality in subjects with impaired glucose tolerance or diabetes: results from the 11 year follow-up of the Paris Prospective Study[END_REF] and mathematical models, have shown a good correlation with the reference standard in non-diabetic patients [START_REF] Ascaso | Diagnosisn Insulin resistance by simple quantitative methods in subjects with normal glucose metabolism[END_REF].

OBJECTIVE

This study evaluates the association between an indirect measure of insulin resistance and diabetic macular edema in type 2 diabetes mellitus patients, specifically the cystic type, and assesses the effect of exogenous insulin treatment on macular edema. Determination of sample size was based on the normal asymptotic confidence interval for the estimated occurrence of an event in an infinite population, considering the percentage of insulin resistance in the general population [START_REF] Ascaso | Diagnosisn Insulin resistance by simple quantitative methods in subjects with normal glucose metabolism[END_REF] to be 32% (95% confidence interval) to achieve an accuracy of ±10.0%. The calculation was performed with ENE 2.0 software (Biometry Department of GlaxoSmithKline and Statistics Service of the Autonomnous University of Barcelona). At least 84 patients were required to fulfill these conditions, and 90 were ultimately included in the study.

METHODS

Results were analyzed with SPSS, version 15.0. The Student t test for independent samples was used to compare patients with and without insulin resistance. Associations were studied in 2x2 tables with the chi-square and Fisher's exact test. Odds ratios were calculated at a 95% confidence interval (CI).

All patients signed an informed consent form to participate, and the study was approved by the local ethics committee.

RESULTS

A total of 177 eyes from 90 patients with type 2 diabetes were included in the study; 27.1% of eyes were from insulin-resistant patients. There were no differences in age, HbA1c, time of evolution of diabetes, sex, or lens status between the insulin-resistant and nonresistant patients. However, these groups showed some differences related to the treatment received: insulin-resistant patients were more likely to have exogenous insulin therapy (P<0.05; OR=3.8).

The morphologic patterns of diabetic macular edema differed according to whether or not the patients were insulin resistant (P=0.031) (Tables 1 and2, Figure 1). When each morphological pattern was compared with insulin resistance, an association was found between diabetic cystoid macular edema (type II) and resistance (Fisher exact test P=0.007; OR=2.53, 95% CI: 1.52-4.2) (Tables 3 and4). There were no associations between insulin resistance and the other morphologic patterns of diabetic macular edema.

In addition, patients undergoing insulin therapy were found to have a direct association with a diffuse retinal thickening pattern on OCT (type I) (P=0.036; OR=1.4). However, no association was found between insulin therapy and the presence of cystoid macular edema (P=0.78).

DISCUSSION

Direct methods, the reference standard for determining insulin resistance, are difficult to perform and expensive; thus, they are not recommended for epidemiologic or clinical studies [START_REF] Ascaso | Diagnosisn Insulin resistance by simple quantitative methods in subjects with normal glucose metabolism[END_REF]. Insulin resistance was established as fasting insulin levels >16 mU/L in the Paris Study [START_REF] Fontbonne | Hypertriglyceridemia as a risk factor of coronary heart disease mortality in subjects with impaired glucose tolerance or diabetes: results from the 11 year follow-up of the Paris Prospective Study[END_REF], which, to our knowledge, is one of the most important prospective studies on this subject.

The McAuley index has been described as the most sensitive and specific indirect method to evaluate resistance to insulin (9). McAuley et al. define

insulin resistance as a score of <6.3 [START_REF] Mc Auley | Diagnosing Insulin Resistance in the general population[END_REF]. Although these methods have been validated in non-diabetic patients, we have used them in our diabetic population as a simple way to evaluate insulin resistance for the purpose of our study. To provide a stricter definition of resistance, we used both methods-fasting insulin levels and the McAuley index-in the patients studied.

The fact that some of our patients were treated with exogenous insulin may have led to an increase in fasting insulin levels, but we consider this an indirect parameter of resistance. Treated patients need more insulin units per kilo because the hormone is less efficiently, and this results in higher fasting insulin levels. The concept of insulin resistance includes exogenous and endogenous insulin. For this reason, we did not differentiate between exogenous/endogenous insulin levels at the time of blood analysis.

Our results showed an association between insulin resistance and exogenous treatment with insulin. It is reasonable that resistant patients would need higher doses of insulin for metabolic control, and this would require exogenous intake of the hormone. Each patient's eyes were analyzed separately because the presence and type of macular edema was different between eyes in some cases.

The main finding of our study is the differing morphological pattern of macular edema related to insulin resistance and, of course, the association between cystoid macular edema and insulin resistance. As was mentioned above, insulin resistance is a proinflammatory state, and the pattern of edema found in these patients is very similar to the pattern seen in inflammatory diseases such as Irvine-Gass syndrome and uveitis [START_REF] Martin | Retinal phlebitis in the Irvine-Gass syndrome[END_REF]. In fact, the pathogenesis of cystoid edema includes both vasoreactivity and chronic inflammation [START_REF] Ryan | Surgical Retina. 114 Vitreoretinal Interface[END_REF], which we believe may be a result of the patient's insulin-resistant status. In other diseases such as vein occlusion, which are primarily considered vascular events, it has been reported that an inflammatory factor and clinical cystoid macular edema are often present [START_REF] Noma | Vitreous levels of interleukin-6 and vascular endothelial growth factor in macular[END_REF].

To define whether the association we found was only related to resistance or was also due to insulin treatment, we investigated the relationship between insulin and macular edema. The fact that a patient is receiving insulin therapy seems to have no relationship with cystoid macular edema.

However, there was an association with a pattern of diffuse retinal thickening, perhaps a result of the vasoreactive effect of insulin [START_REF] Vassiliki | Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxiainducible factor-1 alpha and VEGF[END_REF][START_REF] Zapata | Blurred Vision and increased macular volume in the introduction of insulin therapy: a prospective study[END_REF].

The potential limitations of our study arise from the design and inclusion criteria. This is a cross-sectional clinic-and hospital-based population study with a broad range of inclusion criteria, a simple "photograph" of our patients that involves no treatment interventions. Therefore, there is the possibility of some bias, such as ongoing treatment for macular edema in the 3 months before inclusion, or OCT reading by only one observer.

Nonetheless, we believe that our cohort provides a close representation of daily practice, with patients consulting at our specialized retinal outpatient clinic and our general ophthalmology department, which implies that patients with and without diagnosed diabetic retinopathy were included. This is also the reason why there are many more patients in the group without edema than in the group of DME.

The sample size was difficult to determine. We knew the prevalence of cystoid macular edema in the diabetic population, but we did not know the prevalence of insulin-resistant patients with macular edema or with diabetes. Our sample size reflects the percentage of resistance in the population, but the number of participants was not determined according to the purpose of the study because there was not enough data in the literature to provide a basis for the calculation.

Association between macular edema pattern and insulin resistance STATEMENT "I Josep Badal ,the Corresponding Author of this article (INSULIN RESISTANCE AND DIABETIC MACULAR EDEMA IN TYPE 2 DIABETES MELLITUS) has the right to grant on behalf of all authors and does grant on behalf of all authors, a licence to the BMJ Publishing Group Ltd and its licensees, to permit this Contribution (if accepted) to be published in British Journal of Ophthalmology (BJO) and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at: (http://bjo.bmj.com//ifora/licence.pdf). Please tick one or more boxes as appropriate: _ I am the sole author of the Contribution. X I am one author signing on behalf of all co-owners of the Contribution. _ The Contribution has been made in the course of my employment and I am signing as authorised by my employer. _ I am a US Federal Government employee acting in the course of my employment. _ I am not a US Federal Government employee, but some or all of my co-authors are. _ I am an employee of the UK Government acting in the course of my employment _ I am not an employee of the UK Government acting in the course of my employment but some/all of my co-authors are.

Competing interest: none declared.

  This is a cross-sectional study performed in a clinic-and hospital-based population, enrolled over a 3-week period in our hospital's general ophthalmology department and dedicated retinal outpatient clinic. The study included all type 2 diabetes patients who agreed to undergo blood sample extraction. Patients who had received treatment for macular edema within the past 3 months and those with other retinal diseases were excluded. The following data were recorded in all cases: age, sex, time of diabetes evolution, HbA1C levels, ophthalmologic and systemic treatment, and lens status. The Early Treatment Diabetic Retinopathy Study (ETDRS) criteria were used to determine whether clinically significant macular edema (CSME) was present or not. In patients with CSME, optical coherence tomography (OCT) (Stratus OCT Humphrey Zeiss Inc, San Leandro, California, USA, software version 4.0) was performed to determine the morphologic patterns of macular edema according to the classification of Kim et al (10). OCT scans were graded by one trained reader according to the predominating morphologic patterns present: type I, diffuse retinal thickening; type II, cystoid macular edema; type III, serous retinal detachment without posterior hyaloidal traction; type IV, posterior hyaloidal traction without traction retinal detachment; and type V, traction retinal detachment. Blood samples were collected from all patients. Insulin resistance was established on a McAuley index of <6.3 and fasting insulin levels of >16 mU/L (8,11).

  Despite these potential limitations, our results indicate that insulin resistance plays a central role in the pathogenesis of macular edema, particularly in the cystoid pattern. Future longitudinal studies, with more accurate inclusion criteria and other methods to evaluate resistance are needed to confirm our findings. It could be of great interest to investigate the association between insulin resistance and local factors of inflammation. In summary, the findings of this study indicate a relationship between insulin resistance and cystoid macular edema that is not related to the use of insulin. Insulin treatment was associated with diffuse macular edema. Prospective, longitudinal studies are needed to evaluate the role of insulin and insulin resistance in diabetic macular edema. edema with central retinal vein occlusion. Ophthalmology. 2009; 116(1):87-93.