MD Anja K Eckstein
email: anja.eckstein@uk-essen.de

Christian Loesch

Diana Glowacka

Matthias Schott

Klaus Mann

Joachim Esser

Nils G Morgenthaler

Christian Lösch

Euthyroid and primarily hypothyroid patients develop

Keywords: Graves ophthalmopathy, Euthyroidism, Hypothyroidism, TSH receptor antibodies

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Licence for Publication

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 sublicences such use and exploit all subsidiary rights, as Set out in our licence.

Introduction

Graves' ophthalmopathy (GO) usually occurs in a close temporal relationship with hyperthyroidism. It is rare in patients with normal thyroid function (euthyroid GO) and in patients with hypothyroid forms of thyroid autoimmune disease (hypothyroid GO). Prevalence of GO in primarily euthyroid and primarily hypothyroid ranges between 1.6 and 8.6%. [START_REF] Bartley | Clinical features of Graves' ophthalmopathy in an incidence cohort[END_REF][START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF][START_REF] Marcocci | Studies on the occurrence of ophthalmopathy in Graves' disease[END_REF][START_REF] Paunkovic | Does autoantibody-negative Graves' disease exist? A second evaluation of the clinical diagnosis[END_REF] Diagnosing GO is usually straightforward, but other diagnoses have to be considered in patients with normal thyroid function and only individual signs of GO such as isolated proptosis; these include cavernous carotid fistula, [START_REF] Lore | Carotid cavernous fistula in a patient with Graves' ophthalmopathy[END_REF] sphenoid meningioma and other tumors [START_REF] Asproudis | A sphenoid en plaque meningioma aggravates exophthalmos in a patient with thyroid ophthalmopathy[END_REF] and lymphoma [START_REF] Boyce | Orbital lymphoma masquerading as thyroid ophthalmopathy[END_REF] which may all also accompany GO itself. [START_REF] Stark | Orbital MALT lymphoma in a patient with Graves' ophthalmopathy: a unique observation[END_REF] Determination of thyroid-specific antibodies contributes to the diagnosis of underlying thyroid disease. [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF] found positive results for thyroid-stimulating immunoglobulin (TSI; 93.8%), a first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII; 18.8%) and a second-generation human TSH-binding inhibitory immunoglobulin (hTBII; 81.3%) in 19 euthyroid patients amongst 1020 with GO. TRAb were detectable by at least one method in all patients. [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF] One quarter of euthyroid patients develop thyroid dysfunction in the later course of the disease. [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF] The objectives of this study were to compare clinical symptoms in primarily euthyroid and primarily hypothyroid patients to those in patients with hyperthyroid GO, and to investigate the levels of thyroid-specific antibodies in patients with euthyroid GO.

Patients and methods

Ethics.

The study was approved by the Medical Ethics Committee of the University Essen, Germany. Written consent to be included in the database and to draw blood was obtained from all participants.

Study design. This was a retrospective observational study on data selected from a GO database started in 2000 which includes findings in 893 consecutive patients treated at the University Hospital of Essen, Germany.

Inclusion criteria. Patient records were included if they:

• Presented within 6-12 months of onset of GO before antiinflammatory treatment Euthyroid patients: Patients, who had TSH and FT4 values within the reference range before and within 6 months after GO onset were assigned to the euthyroid group.

•
Treatment. Hyperthyroidism was treated with antithyroid drug (ATD) therapy for one year.

Hyperthyroid patients, who relapsed were given radioiodine therapy, underwent thyroidectomy, or had a further cycle of ATD for one year, according to their preference. Hypothyroid patients were substituted with L-thyroxine to TSH levels between 0.5 and 1 mU/L. Concerning ophthalmopathy all patients received standard antiinflammatory treatment.

During follow up all patients with clinical activity score (CAS) values > 2 were offered treatment with steroids. For mild and moderate disease, they received an oral regime beginning with about 1.5 mg fluorocortolone/mg body weight. Most of the patients received 100mg as starting dosage. Patients with lower body weight started with 8 days of 90mg or 12 days of 80 mg or 16 days of 70mg per day (according to the weight). Afterwards the dosage was tapered by 10 mg every 4 days. The treatment was finished with 4 days of 5mg. So the whole treatment duration was 44 days (cumulative dosage when starting with 100mg/day was 2.22g). For severe disease (increase/side difference of proptosis ≥3mm, reduced monocular excursions more than 15°, double vision in primary position, reduced vision) i.v. steroids (500 mg/day) were given three times within five days and followed by the same oral regime as above. Orbital irradiation was started if patients had impairment of motility and when clinical activity score increased again after termination of steroids.

Clinical assessment and follow up.

Once the patients were included in the database they were seen in regular intervalls about all every 6-12 weeks usually until the end of the treatment (6 months of inactivity/no further ocular surgery). The control intervals were determined by the activity and severity of GO. At every visit the following were assessed: visual acuity, lid width, downward movement of the lids (mm), proptosis (mm), inflammatory signs (redness and swelling of lids and conjunctiva), eye motility (monocular excursions). If visual acuity was reduced, the cornea was stained with fluoresceine, and if slit-lamp and fundus examination showed normal anatomy, optic nerve function was assessed with visual evoked potentials.

The clinical activity score of GO (CAS) was estimated according to Mourits et al. (1991). [START_REF] Mourits | Clinical activity score as a guide in the management of patients with Graves' ophthalmopathy[END_REF] The severity of the eye disease was estimated with a modified NOSPECS classification as described elsewhere. [START_REF] Eckstein | Thyrotropin receptor autoantibodies are independent risk factors for Graves' ophthalmopathy and help to predict severity and outcome of the disease[END_REF] Patients where seen by an endocrinologist at regular basis if necessary all every 4 weeks and thyroid treatment was given according to fT4 and TSH levels.

Measurement of thyroid specific antibodies. TSH-receptor autoantibodies (TRAb) were

measured at the first visit and at the regular control intervalls during follow-up with a secondgeneration TSH-binding inhibitory (TBII) assay based on the human recombinant TSHreceptor (TRAK human LIA®, B.R.A.H.M.S AG, Hennigsdorf/Berlin, Germany). [START_REF] Costagliola | Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease[END_REF] This assay is calibrated in international units (IU), based on the WHO reference standard MRC 90/672. The 50% inhibition of tracer binding corresponds to 7-8 IU/L. Values ≥ 1.5 IU/L (about 10% tracer binding) were regarded as positive, values between 1 and 1.5 IU/L as borderline, and values < 1.0 IU/L as negative, as described in detail elsewhere. [START_REF] Schott | Detecting TSH-receptor antibodies with the recombinant TBII assay: technical and clinical evaluation[END_REF]

Results

Patients and epidemiological findings. Evaluable records were available from 182 patients: 143 hyperthyroid (125 women and 18 men), 28 euthyroid, and 11 hypothyroid patients (31 women and 8 men). Concerning the whole database the prevalence of patients with euthyroidism or hypothyroidism at manifestation of GO was 4.2%.

Patients were followed up for at least 12 months after GO onset, with a median of 36 months for the hyperthyroid patients and 27 months for the euthyroid and hypothyroid patients.

Eighty-eight (61.5%) of the hyperthyroid and 21 (53.8%) of the euthyroid and hypothyroid patients were smokers. The median age of the hyperthyroid patients was 49 years and was 51 years (33-84) in the euthyroid and hypothyroid patients. None of these differences were statistically significant. Patient and disease characteristics are given in table 1. Clinical signs in all patients were assessed before antiinflammatory therapy or 6-12 months after the onset of GO in the absence of antiinflammatory treatment. At the time of assessment according to the fT4 values most of the patients were euthyroid (94% of the primarily hyperthyroid patients, 97% of the primarily hypothyroid/primarily euthyroid patients, n.s.).

Ophthalmological

Euthyroid and hypothyroid patients were similar and developed significantly less severe GO symptoms (NOSPECS score 5.7 versus 4.4; p=0.03) and a less active GO (CAS score 5.2 versus 3.9; p=0.002) than hyperthyroid patients. The frequency of the typical manifestations of GO in both groups are displayed in figure 2. Euthyroid and primarily hypothyroid patients had significantly less soft tissue inflammation (82% versus 96%, p=0.007); and if soft tissue inflammation was already present, did not worsen so severely (figure 2C). None of the euthyroid or hypothyroid patients developed optic neuropathy, while 10 (7%) primarily hyperthyroid patients did. Median monocular excursions were significantly (p=0.04) less reduced in the euthyroid and hypothyroid GO patients (median 35°) than in the hyperthyroid patients (median 30°). The mean Hertel values were very similar in both groups, but there was a significantly higher side difference between both eyes (1.0 mm [hyperthyroid patients] versus 1.9 mm [euthyroid and hypothyroid patients]; p=0.01). Significantly more euthyroid and hypothyroid patients (23%) developed very asymmetric GO with a proptosis difference of ≥ 3 mm (p=0.004) (figure 2B).

Both patient groups received equal amounts of steroids, but irradiation was necessary in only 38% of the euthyroid and hypothyroid patients in comparison to 68% of the hyperthyroid patients (p=0.03).

Endocrinological findings.

The mean thyroid volume was 13 ± 7.6 mL (2-36) in euthyroid and hypothyroid patients and was significantly smaller than in hyperthyroid patients (30 ± 22 ml (8-102).

TRAb levels 6 months after GO onset were significantly higher in hyperthyroid patients (median: 8.6 IU/L) than in euthyroid and hypothyroid patients (median: 2.2 IU/L; p=0.02) (figure 3). Median TPOAb levels 6 months after GO onset were not significantly different: 262 IU/L in hyperthyroid patients and 100 IU/L in euthyroid and hypothyroid patients.

Six months after the first symptoms of GO, only 27 (69%) of the euthyroid and hypothyroid patients had positive findings for TRAb, whilst findings were positive in 134 (94%) hyperthyroid patients. This difference was statistically significant (p<0.0001). Regarding TRAb and TPOAb together still 10 out of 39 (25%) euthyroid and hypothyroid patients were negative for both antibodies in comparision to only 8 out of 143 (5.6%) hyperthyroid patients (p=0.0008).

During the follow up 6 of the euthyroid patients (20.7%) developed hypothyroidism which was substituted with L-thyroxine. Two patients (6,8%) became hyperthyroid and received ATD.

Among the hyperthyroid patients, following ATD treatment, 29 patients (20.3%) went into thyroid disease remission, while 114 patients (79.7%) needed definitive thyroid therapy (radioiodine therapy or thyroidectomy).

Discussion

The clinical signs and symptoms of GO in primarily euthyroid and hypothyroid patients differ from those in primarily hyperthyroid patients: primarily euthyroid and hypothyroid patients show less soft tissue involvement and more asymmetric disease, and the clinical manifestations are less marked. Unilateral GO has often been reported in case reports of primarily euthyroid patients. [START_REF] Lawton | Exclusion of dysthyroid eye disease as a cause of unilateral proptosis[END_REF][START_REF] Lumera | Unilateral Graves Orbitopathy: a case control and retrospective follow up study[END_REF][START_REF] Perrild | The differential diagnostic problems in unilateral euthyroid Graves' ophthalmopathy[END_REF] The diagnosis of GO is considerably impeded by monosymptomatic and asymmetric manifestations, especially in primarily euthyroid patients, and diagnostic imaging is indicated in all such patients. [5-7, 15, 16] There is a close temporal relationship between the onset of hyperthyroidism and the onset of GO, but 4%-18% [START_REF] Bartley | Chronology of Graves' ophthalmopathy in an incidence cohort[END_REF][START_REF] Wiersinga | Temporal relationship between onset of Graves' ophthalmopathy and onset of thyroidal Graves' disease[END_REF] of the patients with Graves disease develop thyroid dysfunction after the onset of GO, most within one year of the onset of eye symptoms. Regular thyroid function tests are therefore indicated in primarily euthyroid patients. About one quarter of primarily euthyroid patients will develop thyroid dysfunction within 4 years. Predictors of this were shown to be suppressed TSH and positive TRAb. [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF] Other minor abnormalities (uneven uptake and the presence of hot or warm lesions), which indicate thyroid involvement in primarily euthyroid patients with GO are seen in the scintigram. [START_REF] Kasagi | Scintigraphic findings of the thyroid in euthyroid ophthalmic Graves' disease[END_REF] Thyroid antibodies are specific indicators for thyroid autoimmunity. Positive TRAb levels confirmed the diagnosis of GO in 69% of the euthyroid and hypothyroid patients. Taking TRAb and TPOAb together the diagnoses of thyroid associated ophthalmopathy could be confirmed even in 75% of the patients. In former studies the long-acting thyroid stimulator protector (LATS) has also been used as indicator for thyroid autoimmunity in euthyroid patients with normal-sized glands [START_REF] Solomon | Identification of subgroups of euthyroid graves's ophthalmopathy[END_REF]. A sensitive test for TRAb in euthyroid patients is necessary because titres are much lower than in primarily hyperthyroid patients. The rate of detection of TBII in euthyroid patients in the second-generation assay has been shown to be significantly higher than that with the first-generation assay (81% versus 18%). [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF] Thyroid-stimulating immunoglobulin (TSI) measurement has been reported to be a useful and possibly more sensitive marker of euthyroid GO than TBII measurement in Asians. [START_REF] Khoo | Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels[END_REF][START_REF] Watanabe | Euthyroid Graves' disease showing no thyroid abnormalities except positive thyroid-stimulating antibody (TSAb): two case reports[END_REF][START_REF] Kazuo | Value of thyroid stimulating antibody in the diagnosis of thyroid associated ophthalmopathy of euthyroid patients[END_REF] This is also the case for primarily hyperthyroid patients. [START_REF] Khoo | The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy[END_REF] Because TSI are less prevalent in white patients, TSI measurement may not be an appropriate predictor of GO in these patients. [START_REF] Gerding | Association of thyrotrophin receptor antibodies with the clinical features of Graves' ophthalmopathy[END_REF][START_REF] Eckstein | Clinical results of anti-inflammatory therapy in Graves' ophthalmopathy and association with thyroidal autoantibodies[END_REF] It must, however, be borne in mind that these results came from different laboratories and that the bioassays used in Asia may have been more sensitive.

Legends for table and figures

Characteristic

Hyperthyroid patients

Euthyroid and hypothyroid patients p-values

  Were followed up for at least 12 months after onset of GO/ until they reached inactivity • Had TRAb and TPOAb titres available 6 months after GO onset • Control group of patients with primarily hyperthyroidism: consecutive patients out of the years 2003 and 2004 in the registry Grouping of the patients. Hyperthyroid patients: Patients, who developed clinical or subclinical hyperthyroidism (elevated/normal fT4 and suppressed TSH) before or within 6 months after the onset of GO were assigned to the hyperthyroid group. Hypothyroid patients: Patients, who developed clinical or subclinical hypothyroidism (reduced/normal fT4 and elevated TSH) before or within 6 months after the onset of GO were concerned as hypothyroid.

  TPOAb were measured in a competitive luminescence immunoassay (B.R.A.H.M.S anti-TPOn LIA B.R.A.H.M.S AG). The threshold for positivity was ≥ 60 U/mL. Outcome measures. Clinical signs in all patients were assessed before antiinflammatory therapy or 6-12 months after the onset of GO in the absence of antiinflammatory treatment. The following parameters were compared among the groups: • Age • Smoking behaviour • Thyroid volume • TBII levels 6 months after first symptoms of GO • TPO Ab levels 6 months after first symptoms of GO • Prevalence of positive TBII 6 months after first symptoms of GO • Prevalence of negative TBII and TPO Ab 6 months after first symptoms of GO • CAS • NOSPECS • Proptosis (mean/side difference) • Upgaze (monocular excursion) • Percentage of patients who had to receive steroid therapy • Percentage of patients who had to receive orbital irradiation Statistical analysis. Group comparisons were tested for statistical difference with Fisher's exact test and with the Mann Whitney Test (Graph pad prism 3.03 software). Fisher's exact test was used for cross tables of patient group vs binary variable (smoking status, dichotomized postive TBII level, dichotomized negative TBII and TPO Ab, dichotomized relative Hertel difference, steroid therapy and orbital irradiation). The number of patients in both groups was compared with an exact Chi-Square-Test. Because of the non-normality of the remaining variables the Mann-Whitney-Test was applied (age, thyroid volume, TBII levels, TPO Ab levels, CAS score, NOSPECS score, Hertel difference, degrees of upgaze, soft tissue inflammation). A p-value less than 5% was regarded as significant.

  findings. Representative patient examples are given in figure 1. All three patients show the typical asymmetric unilateral manifestation of GO in euthyroid and primarily hypothyroid patients. The diagnosis in the first patient was straightforward (1A-1C) because of the typical inferior rectus and levator palpeprae involvement and the soft tissue inflammation. Although thyroid-specific antibody levels in the borderline range had been measured once, this patient had not developed thyroid abnormality after 19 months of followup. The patient in 1D-1F was referred for biopsy with a suspected diagnosis of lymphoma with normal thyroid function. Closer thyroid work-up revealed positive TPOAb (168 U/mL) and TRAb in the borderline range. The patient developed hypothyroidism during follow-up, and lid lengthening was done after 18 months of follow-up. The euthyroid patient in 1G-1K developed unilateral proptosis which progressed over one year, despite steroid treatment. TRAb were 2.4 IU/L at diagnosis and undetectable after one year. Repeated CT scans revealed only an increase in adipose tissue behind the eye. The patient was transnasaly decompressed after 3 years follow-up.

Table 1 :

 1 Patient and disease characteristics.

Figure 1 :

 1 Figure 1: Representative examples of euthyroid and hypothyroid patients: A: enface photograph of a 46 year old patient with severe left unilateral GO with soft tissue inflammation, proptosis and marked impairment of motility. Coronary (B) and transverse (C) MRI scans show thickening and inflammation, especially of the inferior rectus and levator

Figure 2 :Figure 3 :

 23 Figure 2: A: Clinical symptoms of patients with GO and primary hyperthyroidism (black) in comparison to patients with GO and euthyroid or primarily hypothyroid disease (grey). B: Percentage of patients with a proptosis difference of ≥3 mm. C: intensity of soft tissue inflammation

Figure 1 Figure 2 Prevalence

 12 Figure 1

Table 1 :

 1 Patient and disease characteristics

	Number of patients [M/F]	143	39	n.s.
		[18/125]	[9/31]
	Age (years, median, [range])	49	51	n.s.
		[21-79]	[33-84]
	Nonsmoker/smoker	55/88	18/21	n.s.
	Follow up after first symptoms of	36	27
	GO (months, median [range])	[18-73]	[12-79]
	Thyroid volume	23	14	p<0.0001
	(mL, mean [range])	[8-102]	[2-36]
	TBII levels 6 months after first	8.6	2.2	p=0.02
	symptoms of GO (IU/l, median [range])	[<1.5 ->40.0]	[<1.5 ->40]
	Prevalence of positive TBII 6	134 [94%]	27 [69%]	p<0.0001
	months after first symptoms of GO
	(Number [%] of patients)
	Prevalence of negative TBII and	8 [5.6%]	10 [25%]	p=0.0008
	TPO Ab 6 months after first
	symptoms of GO
	(Number [%] of patients)
	TPO Ab levels 6 months after first	262	100	n.s.
	symptoms of GO (U/ml, median [range])	[<60 ->3000]	[<60 ->3000]
	CAS score before antiinflammatory	5.2 ± 2.34	3.9 ± 2.1	p=0.002
	treatment
	NOSPECS score before	5.7 ± 3.1	4.4 ± 2.4	p=0.03
	antiinflammatory treatment
	Hertel (mean±SD)	17.4 ± 3.2	17.3 ± 3.0	n.s.
	R-L difference (mean±SD)	1.0 ± 1.0	1.9 ± 1.8	p=0.01
	% Difference ≥ 3 mm	4.8%	23%	p=0.004
	Upgaze (monocular excursion)	30	35	p=0.04
	(median [range])	[5-40]	[5-40]
	Steroid therapy	124 [87%]	31 [79%]	n.s.
	Number [%] patients)
	Orbital irradiation	98 [68%]	15 [38%]	p=0.03
	(Number [%] patients

M/F=male/female; SD= standard deviation; GO=Graves ophthalmopathy

Competing Interest:

None declared.


Maurizio Battaglia Parodi

MD Pierluigi Iacono

MD Giuseppe Ravalico

M Battaglia Parodi

Fundus Autofluorescence in Subfoveal Choroidal Neovascularization Secondary to Pathologic Myopia

Keywords: Fundus autofluorescence, Choroidal neovascularization, Retinal pigment epithelium, Photodynamic therapy, Pathologic myopia

PURPOSE: to describe the fundus autofluorescence (FAF) characteristics of choroidal neovascularization (CNV) associated with Pathologic Myopia (PM), and their modification after photodynamic therapy (PDT). DESIGN: Open-label, prospective, interventional case series. METHODS: Forty-two patients affected by subfoveal CNV in PM underwent PDT with a 24-month follow-up. Each patient underwent an ophthalmological examination every three months, including FAF and fluorescein angiography. FAF distribution was qualitatively evaluated at the CNV site, around the CNV and outside the area affected by CNV.

RESULTS: CNV at baseline showed high FAF signal with uniform distribution, or with some spots of low FAF internally, in 64% and 36% of cases, respectively. At the 3-month control after PDT, the CNV retained the same response, but a round halo of increased signal extending beyond the site of the PDT application was detectable around the CNV. At the end of the follow-up, a high or a low FAF signal was detected in 40% and 60% of cases, respectively. CONCLUSION: CNV secondary to PM shows a specific, high signal, FAF pattern. A round halo of increased FAF signal surrounding the CNV was detectable after PDT application, whereas a FAF signal progressive reduction was visible at and around the CNV site from the sixth month. A high FAF signal at the CNV site is associated with an improved visual acuity outcome at the 2-year follow-up. Further studies to correlate the morphological and functional features are advisable, especially by means of microperimetric analyses and with a longer-term follow-up.

INTRODUCTION

Fundus autofluorescence (FAF) is determined mainly by the lipofuscin component within the retinal pigment epithelium (RPE) cells. [START_REF] Delori | In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics[END_REF] Lipofuscin originates predominantly from the incomplete digestion of outer segments of photoreceptors and accumulates progressively with age. [START_REF] Feeney | Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies[END_REF] The role of RPE lipofuscin in the pathogenesis of Age-related Macular Degeneration (AMD) has been studied chiefly by means of autofluorescent images, especially as regards geographic atrophy and choroidal neovascularization (CNV). [START_REF] Holz | Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration[END_REF][START_REF] Dandekar | Autofluorescence imaging of choroidal neovascularization due to age-related macular degeneration[END_REF][START_REF] Mcbain | Fundus autofluorescence in exudative age-related macular degeneration[END_REF][START_REF] Framme | Fundus autofluorescence before and after photodynamic therapy for choroidal neovascularization secondary to age-related macular degeneration[END_REF] As yet there are no data concerning either the distinguishing characteristics of the FAF pattern in Pathologic Myopia (PM) or CNV, one of its most feared complications. Myopic CNV is characteristically a type-two CNV, located between the sensory retina and RPE, and is coated with a double RPE layer. [START_REF] Grossniklaus | Clinicopathologic correlations of surgically excised type 1 and type 2 submacular choroidal neovascular membranes[END_REF] RPE modifications, as evidenced by lipofuscin alterations and visualizable by means of FAF, may help provide a better picture of the clinical characteristics and prognosis of CNV secondary to PM.

The aim of the present study is to describe the FAF characteristics of CNV associated with PM, and their modification after photodynamic therapy (PDT).

METHODS

The study prospectively enrolled patients referred to Trieste Eye Clinic between January 2003 and January 2006 with a diagnosis of subfoveal CNV in PM. The research adhered to the tenets of the Declaration of Helsinki, and investigational review board approval was obtained. Each patient was informed of the purpose of the research and provided signed consent to all procedures. Inclusion criteria were: CNV secondary to PM, subfoveal CNV (including both CNV under the geometric centre of the foveal avascular zone and CNV involving the fixation point), evidence of classic CNV on FA, greatest linear dimension under 5400 microns, best-corrected visual acuity (BCVA) from 20/200 to 20/40 on ETDRS charts, and symptom duration of under one month. PM was defined as an eye requiring a distance correction of at least -6.0 diopters (spherical equivalent) and/or axial length was at least 26.5 mm, as well as fundus abnormalities -other than subretinal CNV -compatible with PM, including one or more of the following features: lacquer cracks, peripapillary atrophy, macular geographic atrophy, diffuse fundus pallor.

Exclusion criteria were: evidence of any condition other than PM associated with CNV, any significant ocular disease that had compromised or could compromise vision in the study eye, active hepatitis or clinically significant liver disease, porphyria or other porphyrin sensitivity, pregnancy, intraocular surgery within the last 2 months or capsulotomy in the study eye within the last month, and previous laser photocoagulation.

Each patient underwent an ophthalmological examination, including BCVA on standard ETDRS charts and fluorescein angiography (FA) at baseline, every 3 months over a planned follow-up of 24 months. Subfoveal CNV had to be treated with PDT, adhering strictly to VIP guidelines. FAF was performed using a Heidelberg Retina Angiograph (Heidelberg Engineering, Germany), averaging series of 9-15 digital images to reduce noise. The distribution of FAF was evaluated at the site of the CNV, around the CNV and outside the area affected by the CNV (background autofluorescence). FAF was defined as low (signal lower than background), high (signal higher than background) or unchanged compared with background (signal indistinguishable from background). Distribution of FAF outside the area affected by the CNV (background autofluorescence) was classified as homogeneous, reticular (ill-defined small areas of diminished autofluorescence surrounded by areas of greater autofluorescence), focal (focal areas of increased autofluorescence) or mixed (focal and reticular patterns of autofluorescence).

RESULTS

Forty-two out of 46 patients affected by subfoveal CNV in PM were recruited. Four patients were excluded because they had already undergone PDT in other centres. The mean number of PDT sessions was 2.5 (range: 1-4) in the first year and 1.5 (range: 1-3) in the second year. All the eyes showed a newly-diagnosed subfoveal CNV with classic aspect, on the basis of FA findings.

In all the eyes studied, there was a high FAF signal in correspondence with the CNV site at baseline (Fig. 1). More specifically, two subgroups could be singled out: 27 eyes (64%) showed a high FAF signal with uniform distribution (Fig. 1, 2), whereas 15 (36%) eyes displayed an irregular pattern characterized by high FAF signal, containing spots of low FAF inside (Fig. 3). A fine hyperautofluorescent signal bordered the CNV at least in part in all the cases, which was found on FA to consist of a thin area of blocked fluorescence around the CNV. The FAF pattern appeared to be homogeneous outside the CNV.

At the 3-month control after the PDT treatment, the CNV retained the same high autofluorescent response, displaying a distinct demarcation. A round halo of increased signal extending beyond the site of the PDT application was detectable surrounding the CNV in all the cases (Fig. 1,2). The remaining posterior pole showed a homogeneous pattern of FAF.

The subsequent follow-up examinations revealed that the high autofluorescent signal corresponding to the CNV site gradually faded away (Fig. 1,2). In particular, at the end of the 24-month followup, 17 CNVs (40%) showed a uniformly high FAF response, whereas 25 CNVs (60%) revealed a central low FAF signal (Fig. 3). This latter group was composed of all the 15 CNVs displaying spots of low FAF and by 10 CNVs characterized by a high FAF signal at baseline. The halo of high autofluorescence neighboring the CNV was still visible over the whole follow-up, even though it displayed a partial signal reduction at the 24-month examination.

Overall, a progressive reduction of the FAF signal was visible at and around the CNV site from the 6 th month, probably due to the progressive atrophic RPE changes.

Analysis of the correlation between BCVA and intensity of FAF signal at baseline examination revealed the absence of any statistically significant difference, when the subgroup characterized by high FAF signal was compared with the subgroup with irregular FAF. By contrast, a statistically significant difference was registered at the end of the follow-up between BCVA values in the eyes retaining high FAF signal and those displaying irregular FAF signal (p<0.001) (Fig. 4). Moreover, at the end of the follow-up a statistically significant deterioration of BCVA was registered in the subgroup with irregular FAF (p= 0.015), whereas BCVA did not reveal any statistically significant change in the subgroup retaining high FAF signal. A statistically significant difference (p<0.001) was observed within the subgroup of eyes characterized at baseline by a high FAF signal and comparing BCVA in eyes retaining the same high FAF pattern with that of the developing irregular FAF pattern.

DISCUSSION

Being a type 2 CNV, situated between the sensory retina and RPE and entirely coated by RPE, myopic CNV has specific histopathological features. PM-related CNV is characterized by an RPE layer on its inner surface and an inverted RPE layer on its outer surface, creating a twofold RPE layer. [START_REF] Grossniklaus | Clinicopathologic correlations of surgically excised type 1 and type 2 submacular choroidal neovascular membranes[END_REF] All these histopathological characteristics are to be borne in mind when analyzing the aspects found on FAF. Myopic CNV generally displays a high autofluorescent signal at its site, to be explained by the presence of a double RPE layer wrapping the CNV. In this regard, this FAF pattern is completely different from that typical of classic CNV secondary to AMD. Indeed, this form of classic CNV shows a low autofluorescent signal, probably due to the blockage of the autofluorescence caused by the presence of the CNV in the subretinal space. [START_REF] Mcbain | Fundus autofluorescence in exudative age-related macular degeneration[END_REF] Nevertheless, from the baseline examination, 36% of eyes showed some spots of low FAF signal within the high FAF response of the CNV. These hyporeflective spots can be explained as areas of damaged RPE.

The presence of a fine margin of high FAF bordering the CNV at baseline can be interpreted as being determined by the RPE cell proliferation around the CNV. [START_REF] Miller | The role of retinal pigment epithelium in the involution of subretinal neovascularization[END_REF] The most intriguing feature is the development of a round halo with high autofluorescence encircling the CNV, detectable after the first PDT administration. This is a newly reported feature and is not detectable on FA. This halo may be directly linked to PDT application, but interestingly the visible limits of the circle lie outside the area exposed to the laser light. The PDT-induced production of lipofuscin within the RPE cells may explain the development of such a halo. It is noteworthy that the effect on the RPE extends beyond the boundary of the area directly affected by the laser.

Moreover, a prolonged follow-up reveals a gradual reduction of the autofluorescent signal, detectable both at the CNV site and in the surrounding areas, probably owing to the atrophic RPE changes.

The effect on the RPE of PDT administered for PM-related CNV has been previously analyzed on FA [START_REF] Parodi | Retinal pigment epithelium changes after photodynamic therapy for choroidal neovascularization in pathological myopia[END_REF] , revealing progressive RPE alterations, such as accentuation of the pigmentary zone surrounding the CNV and RPE depigmentation and atrophy. [START_REF] Parodi | Retinal pigment epithelium changes after photodynamic therapy for choroidal neovascularization in pathological myopia[END_REF] All these features have a correlation with FAF, manifested by the gradually decreasing autofluorescent signal, both at the CNV site and at the adjacent retina. RPE involvement due to PDT is a matter of debate. 10,[START_REF] Parodi | The origin of the hypofluorescent spot after photodynamic therapy[END_REF] Even though no ultrastructural study has specifically targeted RPE cell alterations induced by PDT, speculative deductions suggest that PDT acts on RPE, leading to clinical and angiographic results.

The presence of normal or near-normal FAF over areas of CNV in AMD has been correlated to a normal or near-normal RPE, resulting in a better functional prognosis [START_REF] Dandekar | Autofluorescence imaging of choroidal neovascularization due to age-related macular degeneration[END_REF] , and our results support this conclusion. The comparison between the subgroup retaining a high FAF signal and the subgroup developing a low signal at the end of the follow-up revealed a statistically significant difference in the BCVA. This finding suggests that the initial FAF pattern can provide information regarding the final functional outcome after the PDT application.

The present study cannot explain the reasons underlying the different FAF pattern detectable at baseline. The FAF pattern may reflect a number of features, including RPE cell viability, CNV duration, presence of fluid or blood or biochemical changes that are not directly quantifiable yet.

We acknowledge that this study has important limitations, especially regarding the lack of an untreated control group. The small sample of patients did not allow a stratification of BCVA values in the subgroups. Moreover, the study aims at a qualitative rather than a quantitative analysis of the FAF distribution and looks at the identification of changes in autofluorescence patterns. It is possible that a quantitative evaluation of the FAF will reveal more important details.

In conclusion, CNV secondary to PM exhibits a specific, high-signal, FAF pattern. A round halo of increased FAF signal surrounding the CNV is detectable after PDT application. Overall, a progressive reduction of the FAF signal was visible at and around the CNV site from the 6 th month, probably due to the progressive atrophic RPE changes. A high FAF signal at the CNV site is associated with a better visual acuity outcome at the 2-year follow-up. Further studies are advisable to correlate the morphological and functional features, especially by means of microperimetric analyses and including a longer-term follow-up. 1 0 Legend: Statistical analyses showed that there is a statistically significant difference at the end of the follow-up between BCVA values in the eyes retaining a high FAF signal and those displaying an irregular FAF signal (p<0.001). Moreover, at the end of the follow-up a statistically significant deterioration of BCVA was registered in the subgroup with irregular FAF (p= 0.015), whereas BCVA did not reveal any statistically significant change in the subgroup retaining a high FAF signal.

Fig. 1 .

 1 Fig. 1. Top left: FAF of a subfoveal CNV showing a high signal before PDT (the white line

Fig. 2 .

 2 Fig. 2. Top left: FA of a subfoveal CNV at baseline. Bottom left: FAF of the same case showing a

Fig. 3 .

 3 Fig. 3. Top left: FA of a subfoveal CNV at baseline. Top right: FAF of the same case at baseline

Figure 4 .

 4 Figure 4. Mean change of visual acuity (logarithm of the minimum angle of resolution [logMAR]) in the subgroups of patients with choroidal neovascularization showing homogeneously high FAF signal and irregular FAF pattern during the 24-month follow-up.


MD Isabel Lema
email: mariaisabel.lema@usc.es

Tomas Sobrino

Juan A Duran

David Brea

Elio Diez-Feijoo

PhD Tomás Sobrino

MD Juan A Durán

David Brea Bsc

MD Elío Díez-Feijoo

Subclinical keratoconus and inflammatory molecules from tears

Keywords: Keratoconus, Inflammatory molecules, tears. Word count: 2683

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Keratoconus (KC) is a primary corneal ectasia, generally bilateral and progressive, with a conical shape as a result of the thinning of the stroma. This thinning induces irregular astigmatism, leading to mild to marked impairment in the quality of vision. [START_REF] Krachmer | Keratoconus and related noninflammatory corneal thinning disorders[END_REF] KC has its onset at puberty, and it progresses until the third or fourth decade of life. [START_REF] Rabinowitz | Keratoconus[END_REF] In most cases, KC initially affects only one eye. As ectasia develops, it may remain restricted to one eye, but more frequently progresses to both. The second eye may develop KC or remain in an unchanging state referred to as the "forme fruste" or subclinical KC. [START_REF] Maguire | Identifying progression of subclinical keratoconus by serial topography analysis[END_REF] Currently, reported frequencies of unilateral KC, as determined by using computerized videokeratography techniques, range from 0.5% to 4%. [START_REF] Rabinowitz | Videokeratography of the fellow eye in unilateral keratoconus[END_REF][START_REF] Holland | Unilateral keratoconus: incidence and quantitative topographic analysis[END_REF] In a recent study, in which 85 unilateral KC patients were monitored over a long period (6 months to 8 years), authors have found that (35.3%) of initially healthy eyes, developed KC during the follow-up period, and (83.3%) of these developed KC within the first 6 years after the initial KC diagnosis. Likewise, the risk of KC onset in initially healthy eyes was higher for those with greater inferior-superior asymmetry (I-S asymmetry). Authors have estimated that over a 16-year period follow-up, about 50% of initially healthy contralateral eyes will develop KC. [START_REF] Xiaohui | Longitudinal study of the normal eyes in unilateral keratoconus patients[END_REF] Unilateral KC can be studied to provide insight into the pathogenesis of the disease. In fact, given that KC may arise without biomicroscopic signs, examination of the initially healthy eye may be useful for the detection of subclinical changes and progression of the disease. [START_REF] Wilson | Screening for corneal topographic abnormalities before refractive surgery[END_REF] There is increasing evidence that supports the fact that KC is an inherited genetic disorder. If this were the case, it would be expected to express itself as a binocular condition, even though variable expression of the gene may show differing levels for each eye. The videokeratoscopy has shown almost every case of apparent monocular KC to be different expressions of the condition in the two eyes. [START_REF] Phillips | Can true monocular keratoconus occur?[END_REF] Videokeratoscopes enable the detection of small changes in the corneal topography, which is esential for the detection and monitoring of KC. [START_REF] Rabinowitz | Videokeratographic indices to aid in screening for keratoconus[END_REF][START_REF] Lee | Clinical detection of unilateral keratoconus[END_REF] Orbscan II, that uses a slit scanning procedure to map the anterior and posterior corneal surface, may help to identify frustrated or subclinical forms of KC in a short time. [START_REF] Rao | Role of Orbscan II in screening keratoconus suspects before refractive corneal surgery[END_REF] The cornea is part of an integrated system -the ocular surface -which contains specific and non-specific immune molecules. Tissue degradation in thinning disorders, such as keratoconus, involves the expression of inflammatory mediators, such as proinflammatory cytokines, cell adhesion molecules and matrix metalloproteinases. [START_REF] Chandler | Ocular surface immunology[END_REF][START_REF] Bonini | Inflammatory immune-associated diseases of the cornea[END_REF][START_REF] Zhou | Expression of degradative enzymes and protease inhibitors in corneas with keratoconus[END_REF][START_REF] Collier | Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas[END_REF]. Moreover, previous studies from our group have demonstrated that the levels of proinflammatory cytokines [interleukin-6 (IL-6) and the tumor necrosis factor alpha (TNF-α)] and matrix metalloproteinase-9 (MMP-9) are significantly increased in the tears of patients with KC. [START_REF] Lema | Inflammatory molecules in the tears of patients with keratoconus[END_REF] The purpose of this study was to measure levels of proinflammatory cytokines (IL-6 and TNF-α) and MMP-9 in the tears of both eyes from asymmetric KC patients, and to analyze the possible difference between subclinical and KC eyes and normal controls, with a view to contribute to our understanding of whether inflammatory molecules may enhance the progression to KC.

MATERIALS AND METHODS

Patients

We have designed a prospective, cross sectional study in which 30 asymmetric KC patients (30 KC and 30 subclinical KC eyes) and 20 control subjects ( 1 eye) were enrolled. Asymmetric KC patients (70% male; mean age, 27.1 ± 8.1 years) were selected from a database of 385 KC patients at the Instituto Galego de Oftalmoloxía, Santiago de Compostela, Spain, which makes up 7.8% of the sample. We studied both eyes from each patient. Fourteen patients wore rigid gas-permeable (RGP) contact lenses in the keratoconic eye. In addition, 3 of them wore a hydrogel contact lens in the fellow eye. Patients were asked not to wear their contact lenses for a week prior to the study. Controls (47.8% male; mean age, 22.6 ± 6.6 years) were myopic subjects who requested contact lenses, without previous history of keratoconus or eye rubbing, who presented normal topographic patterns. All patients were expressly cited for the purposes of this study, and all examinations were performed by the same researcher between October 2005 and June 2006. Protocol was approved by the Hospital's Research Ethics Committee, and informed consent was given by all patients and control subjects. Data collected included gender, age, patient´s ocular history, medical history (allergy, eye rubbing), and family history. Inclusion criteria were; 1) Asymmetric KC patients (1 KC eye and 1 subclinical KC eye).

Rabinowitz/McDonnell criterion was used for the KC diagnosis. [START_REF] Rabinowitz | Keratoconus[END_REF] According to the videokeratographic guidelines proposed by Rabinowitz and McDonnell, it is possible to classify an eye as subclinical keratoconus if the simulated central corneal power is greater than 47.2 D but less than 48.7 D, with an inferior-superior dioptric asymmetry greater than 1.4 D, but less than 1.9 D. Eyes presenting central corneal curvature exceeding 48.7D, as well as inferior-superior dioptric asymmetry greater than 1.9 D can be classified as "true keratoconus". 2) The distance visual acuity (DVA) of the subclinical KC eye with value of 1.0 (without correction or with spherical and /or cylindrical compensation < 1.50D).

Exclusion criteria included; 1) Previous surgical intervention in the anterior segment, or childhood corneal trauma or disease. 2) Existence of active or systemic inflammation, or ocular disease, or current treatment with systemic or local anti-inflammatory drugs.

Instrumental

Basic examination instruments were a Topcon biomicroscope, Topcon refraction column, and alphabetic Snellen visual acuity test. As specific examination instruments we used an EyeSys Corneal System (EyeSys Laboratories, Texas, USA), and an Orbscan II corneal topographer (Orbtek, Utah, USA).

Procedure

Our protocol collected the following information: elapsed time from diagnosis of KC in the first eye, laterality, itching and rubbing, family history of KC and allergies.

After obtaining the best correction, the biomicroscopy exam was performed to detect signs of KC. The corneal topography study was performed using the EyeSys and the Orbscan topographers. Five quantitative topographic parameters were analyzed: simulated keratometry (K) readings, 3 mm irregularity, 5 mm irregularity, posterior elevation and the thinnest point pachymetry of the cornea.

Tear analysis

Tear samples were obtained by capillary flow as previously described. [START_REF] Lema | Inflammatory molecules in the tears of patients with keratoconus[END_REF] Briefly, tear samples were obtained by capillarity attraction, without nasal stimulation or previous instillation of drugs or vital dyes. We collected 10 μL of tear sample in micropipettes (Disposable Micro Sampling Pipettes, Corning, NY, USA), placing them in microtubes (Micro Titertube natural 845-TP, TTE Laboratories, Hopkinton MA, USA). A new capillary tube was used for each tear sample. Samples were frozen and stored at -80 ºC within 1 hour after collection.

Concentration of cytokines (IL-6 and TNF-α) in tear samples were measured with commercially available quantitative sandwich enzyme-linked immunoadsorbent assay kits (Quantikine, R&D Systems, Minneapolis, MN, USA) and the assays were performed in accordance with the supplier's instructions. MMP-9 levels were determined by commercially available enzyme linked immunoadsorbent assay (ELISA) kits (Biotrack, Amersham Pharmacia Biotech, Buckinghamshire, UK). ELISAs were performed according to the manufacturer's instructions. Samples were diluted 1:20 with sample solvent to a final volume of 300 μl. All determinations were carried out without knowledge of the corresponding clinical data (blind test), and the final results were multiplied by the dilution factor (x20).

Statistical analysis

Descriptive statistical analyses were performed with percentage for categorical variables. Discontinuous variables were expressed as median [quartiles]. Graphic expressions were elaborated by Box and Whisker plots.

Statistical significance for intergroup differences was assessed by the χ2 test for categorical variables. Inflammatory molecular markers values were not normally distributed (Kruskal-Wallis analysis). One-way ANOVA and the Mann-Whitney test were performed for comparison between groups. The Spearman correlation coefficient was used to analyze the statistical significance between quantitative topographic parameters (the thinnest point pachymetry of the cornea) and the concentrations of molecular marker (MMP-9). A value of p < 0.05 was considered to be statistically significant.

RESULTS

Clinical Features

No age-related or sex-related statistical differences were detected between the groups.

The elapsed time from the first diagnosis of KC eye ranged from 1 to 16 years (mean, 5.6 ± 4.3 years). In 22 patients (73.3%) the KC eye was the right one. A family history of KC was reported for 8 patients (26.6%). Eighteen KC patients (60%) reported allergy disease and 15 (50%) a family history of allergy (most commonly to pollen or acaroids).

Nineteen patients (63.3%) reported itchy eyes and, in 69% of them itchiness was more marked in the KC eye. For patients who reported itchy eyes, 58% admitted frequent and vigorous eye rubbing on the KC eye. Three subjects (15%) from the control group reported allergic disease, and no eye rubbing. Mean K 2 was 51.5 ± 7.3 D in the affected eye, versus 43.9 ± 1.4 D in the subclinical KC eye. Astigmatism (dK) was 3.44 ± 2.97 D in the KC eye versus 0.96 ± 1.12 D. Mean of the thinnest point pachymetry was 407.8 ± 59.3 µm in the KC eye, and 499.4 ± 37.6 µm in the contralateral eye. Table 1 shows the quantitative topographic parameters in KC, subclinical KC and control subjects. Patients with asymmetric KC showed significantly higher levels of IL-6 and TNF-α in both eyes. Mean values for IL-6 levels were similar in KC and subclinical KC samples (5.5 [4.9 -6.9] vs. 5.7 [4.5 -6.2] pg/mL, p= 0.131), but significantly higher in relation to control group (2.2 [1.0 -4.1] pg/ml, p<0.0001). Likewise, significant differences were found in TNF-α levels between KC and subclinical KC eyes (5.4 [4.1 -6.8] 1).

Association between levels of MMP-9 and the thinnest point pachymetry of the cornea in KC and subclinical KC eyes was also analyzed. As indicated in Figure 2 Placido disk methods, it may be difficult to detect any changes in the anterior corneal face of the contralateral eye. However, Orbscan reveal a raised posterior face and pachymetry, indicative of incipient KC. Binder has also reported KC cases in which topographic changes in the contralateral eyes were observed after LASIK surgery, suggesting that these cases are in fact bilateral KC with marked asymmetry, instead of true asymmetric KC. [START_REF] Binder | Ectasia after laser in situ keratomileusis[END_REF] It likely seems that in most cases, unilateral KC exists only in the early stages of disease, and that in such cases, asymmetry is simply due to unevenness in disease development. In order to try to corroborate the hypothesis that KC is bilateral in last stage, we performed this study about inflammation molecules from tears, which were previously shown to be elevated in KC patients, with the aim of analyze whether inflammatory molecules may enhance the progression of Keratoconus. [START_REF] Lema | Inflammatory molecules in the tears of patients with keratoconus[END_REF] It seems becoming clear that mediators of inflammation are present in the keratoconus cornea. [START_REF] Zhou | Expression of degradative enzymes and protease inhibitors in corneas with keratoconus[END_REF][START_REF] Smith | Tear film MMP accumulation and corneal disease[END_REF] The presence of collagen degradation products has been reported in the tears of keratoconus patients. [START_REF] Abalain | Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus[END_REF] In the present study, we demonstrate that the levels of studied cytokines (IL-6 and TNF-α) are significantly increased in the tears from both eyes of patients with asymmetric keratoconus. To our knowledge, this is the first study supporting an important increase of proinflammatory markers in the tear film of subclinical keratoconus eyes. However, MMP-9 levels in tears from subclinical KC eyes are not raised, presenting MMP-9 levels similar to those of control eyes.

Preocular tear film contains numerous specific and non-specific immune components, that include cytokines and cell adhesion molecules. [START_REF] Sonoda | Inflammatory cytokine of basal and reflex tears analyzed by multicytokine assay[END_REF][START_REF] Solomon | Pro-and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease[END_REF] Increased levels of these molecules may be a consequence of increased secretion from the corneal epithelium or from another non-corneal cell type. Various cell types, including keratocytes, produce IL-6 in response to stimulation by IL-1 or TNF-α. [START_REF] Planck | Cytokine mRNA levels in rat ocular tissues following systemic endotoxin treatment[END_REF] In recent years, numerous clinical studies of keratoconus support the idea that its pathogenesis involves an inflammatory component. A variety of changes in the ocular surface of keratoconus patients have been found, including reduced corneal sensitivity, increased fluorescein and rose Bengal staining scores, and abnormal impression cytology such as squamous metaplasia and lower globet cell density. [START_REF] Dogru | Tear function and ocular surface changes in keratoconus[END_REF] In the same way, increased enzymatic activity has been demonstrated in the conjunctiva of keratoconus patients. [START_REF] Shen | Lysosomal hydrolase staining of conjunctival impression cytology specimens in keratoconus[END_REF] Overall, it cannot be ruled out that keratoconus originates in events which take place outside the cornea but which are ultimately responsible for the induction of its ectasia.

Association between keratoconus and allergy, [START_REF] Bawazeer | Atopy and keratoconus: a multivariate analysis[END_REF] as well as the role played by eye rubbing in the development of ectasia are well established. Eye rubbing may well contribute to the development of keratoconus by activating inflammatory mediators.

The tear may be a vehicle of some of the pathogenic protagonists of keratoconus, such as IL-6, TNF-α or MMP-9. Our results indicate that the concentration of inflammatory molecules in tears is associated with both eyes from asymmetric KC patients, although TNF-α is significantly higher in the KC eye, respect to the subclinical one. This difference could explain the differences in progression for both eyes, since IL-1β and TNF-α have been shown to up-regulate MMP-9 expression in human corneal epithelial cells. [START_REF] Li | Regulation of MMP-9 production by human corneal epithelial cells[END_REF] Nevertheless, the presence of different classes of enzymes which play a role in the pathological process is not necessarily accompanied by an immediate presentation of corresponding clinical manifestations. [START_REF] Sonoda | Inflammatory cytokine of basal and reflex tears analyzed by multicytokine assay[END_REF] We must also bear in mind that there is the possibility that the changes in these inflammatory mediators may be an epiphenomenon of change in corneal structure. It is likely that keratoconus is a disease with a multivariable origin, in which corneal ectasia results from the degradation of stromal collagen.

Thinning and ectasia of the cornea are suggestive of a degraded extracellular matrix. It has become clear that inflammatory events take place in keratoconus and that KC is a bilateral disease that needs inflammatory conditions to progress along the time. The present study reveals that the increase levels of MMP-9 are associated with the corneal thinning; being the minimal corneal thickness related with increases values of MMP-9 in the clinical KC eye. In light of the present results, we believe that when the KC eye diagnosis is achieved for a patient, especially for young people, there is a risk of progression of this disease.

In conclusion, this study reveals that some cytokines such as IL-6 and TNF-α are overexpressed in the tears of subclinical and Kc eyes in patients diagnosed with unilateral KC. However, the precise role of each of these molecular factors still needs to be defined in further studies. These results indicate that KC cannot be defined as a non-inflammatory disorder and that the pathogenesis of KC progression may involve chronic inflammatory events.

  , a significant correlation was found between the concentration of MMP-9 in keratoconus tears and the thinnest point pachymetry, by simple regression analysis. The Spearman coefficient associated with the concentration of MMP-9 and the thinnest point pachymetry in KC eye was -0.374 (p=0.042) (Figure2A); and -0.172 (p=0.362) for subclinical KC eye (Figure2B).DISCUSSIONDespite extensive basic and clinical studies of Keratoconus in recent years, the precise mechanisms underlying this pathology still remain largely unknown. In this study, we have performed a detailed analysis of both eyes in patients with asymmetric keratoconus (1 eye with classic signs of KC, and the other with subclinical KC). Our results suggest that in such cases the contralateral eye typically shows certain topographic and pachymetric changes, indicative of subclinical KC. These results are in line with a recent controversy about if true monocular keratoconus can occur.[START_REF] Mahon | Can true monocular keratoconus occur?[END_REF] Using

Figure 1

 1 Figure 1 Concentrations of inflammatory molecules in Keratoconus (KC) subclinical (SC-KC) and controls tears. A, Interleukin-6 (IL-6). B, Tumor necrosis factor α (TNF-α). C, Metalloproteinase 9 (MMP-9). *p<0.0001, in relation to the control group; **p<0.0001, in relation to the SC-KC; ***p<0.05, in relation to the SC-KC.

Figure 2

 2 Figure 2Correlation between the concentration of MMP-9 and the thinnest point pachymetry of the cornea in KC (2A) and subclinical KC eyes (2B).

TABLE 1 : QUANTITATIVE TOPOGRAPHIC PARAMETERS.

 1

	TOPOGRAPHIC	KERATOCONUS	SUBCLINICAL KC	CONTROL
	PARAMETERS	(N = 30)	(N = 30)	(N = 20)
	Central K (D)	50.9±6.3	43.6±1.7	42.9±0.9
	K2 (D)	51.5±7.3	43.9±1.4	43.2±1.1
	Astigmatism (D)	3.4±2.9	0.9±1.1	0.6±0.7
	Max. K inferior(D)	53.6±5.9	44.2±1.4	43.3±1.2
	Post.elevation	0.082±0.035	0.032±0.011	0.023±0.007
	Thinnest P (μm)	407.8±59.3	499.4±37.6	534±23.2

Values are expressed in mean (± SD)

Inflammatory mediators

Levels of inflammatory molecules in KC, subclinical KC and controls tears are shown in

Table 2 .TABLE 2 : LEVELS OF INFLAMMATORY MOLECULES FROM TEARS. MOLECULAR MARKER KERATOCONUS (N = 30) SUBCLINICAL KC (N = 30) CONTROL (N = 20)

 22

	IL-6 (pg/mL)	5.5 [4.9 -6.9]*	5.7 [4.5 -6.2]*	2.2 [1.0 -4.1]
	TNF-α (pg/mL)	5.4 [4.1 -6.8]*/***	4.8 [4.2 -6.0]*	1.8 [1.5 -2.3]
	MMP-9 (ng/mL)	59.4 [50.6 -66.1]*/**	7.0 [4.8 -8.6]	6.1 [3.9 -8.3]
	Values are expressed in median [quartiles]
	*p<0.0001, in relation to control group.
	**p<0.0001, in relation to subclinical KC.
	***p<0.05, in relation to subclinical KC.
	IL-6: interleukin 6; TNF-α: tumor necrosis factor α; MMP-9: matrix metalloproteinase-9.

ACKNOWLEDGMENTS

This project has been supported by a grant from the Xunta de Galicia, PGIDIT07PXIB918088PR.

LICENCE FOR PUBLICATION

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 sublicences such use and exploit all subsidiary rights, as set out in our licence (http://bjo.bmj.com/ifora/licence.pdf).

COMPETING INTEREST

None declared.


Dr Katharina E Schmid-Kubista
email: katharina.schmid-kubista@wienkav.at

Ilse Krebs

Birgit Gruenberger

Johann Schueller

Susanne Binder

Systemic bevacizumab (

Keywords: Bevacizumab, Systemic Therapy, Age-Related Macular Degeneration, Placebo BEAT-AMD-Study 1

published or not. The documents may come

Introduction

Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) is one of the major causes of severe vision loss in the elderly in North America and Europe.

[1] AMD threatens the distance as well as the reading acuity and it is already known that the vascular endothelial growth factor (VEGF) plays a major role in the development of CNV. [START_REF] Kliffen | Increased expression of angiogenic growth factors in age-related maculopathy[END_REF] When we began the present study, in June 2005, promising results were published of phase I/II clinical trials after intravitreal injections of the anti-VEGF drugs pegaptanib (Macugen®, Eyetech Pharmaceuticals), [START_REF] Study | Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration[END_REF]4] ranibizumab (Lucentis®, Genentech Inc.), [START_REF] Michels | Ranibizumab therapy for neovascular age-related macular degeneration[END_REF] and systemic treatment with bevacizumab (Avastin®, Genentech Inc.). [START_REF] Michels | Systemic Bevacizumab (Avastin) therapy for neovascular age-related macular degeneration[END_REF] At that time a systemic treatment, which would only need to be administered a few times within a short time, was very tempting, especially for patients with lack of quality of life due to bad visual acuity.

Photodynamic therapy (PDT), [START_REF]Tretament of Age-Related Macular Degeneration with Photodynamic Therapy (TAP) Study Group. Photodynamic therapy of subfoveal choriodal neovascularisation in age-related macular degeneration with verteporfin: oneyear results of 2 randomized clinical trials-TAP report[END_REF][START_REF] Vip Study | Verteprofin therapy of subfoveal choriodal neovascularisation in age-related macular degeneration: two-year results of a randomized clinical trial including lesions with occult with no classic choriodal neovascularisationverteprofin in photodynamic therapy report 2[END_REF][START_REF] Krebs | Reading ability and central visual field after photodynamic therapy[END_REF] in combination with intravitreal injection of triamcinolone acetonide, [10,11] and subretinal surgery [12][13][14] were the common treatments for AMD. These methods were either very invasive or had to be repeated several times over long periods of time in order show some effect. PDT was only performed in eyes with predominantly classic lesions and pigment epithelium detachments (PED) of maximum 50% of the lesion and there was little chance of vision improvement in these eyes. There was no treatment available for larger minimally classic and occult-only lesions or lesions including larger PED. [START_REF] Michels | Systemic Bevacizumab (Avastin) therapy for neovascular age-related macular degeneration[END_REF] Bevacizumab was a new anti-VEGF which showed promising results as a combination therapy with 5-fluorouracil, leucovorin, and oxaliplatin in first-line treatment of metastatic colorectal cancer[15, 16] and as systemic therapy against AMD. [START_REF] Michels | Systemic Bevacizumab (Avastin) therapy for neovascular age-related macular degeneration[END_REF] However, only a case series for AMD treatment did exist. The rationale of the present study was to determine the effect of systemic bevacizumab therapy in subjects with fibrovascular PED, involving the geometric centre of the foveal avascular zone, in comparison to placebo treatment with sodium chloride 0.9%. The main outcome measure was the lesion size, while best corrected visual acuity (BCVA), reading visual acuity (RVA), and macular thickness were additional outcome measures. We believed that a decrease in density of the subfoveal CNV may well lead to a decrease in macular thickness, thereby allowing a better BCVA. Systemic Avastin treatment promised to be a major effort in AMD therapy, although it is mainly administered off-label as intravitreal injection these days. [START_REF] Rosenfeld | Optical coherence tomography findings after intravitreal injection of bevacizumab (Avastin) for neovascular age-related macular degeneration[END_REF][START_REF] Avery | Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration[END_REF][START_REF] Jonas | Visual acuity change after intravitreal bevacizumab for exudative age-related macular degeneration in relation to subfoveal membrane type[END_REF][START_REF] Pedersen | Treatment of choroidal neovascularization using intravitreal bevacizumab[END_REF][START_REF] Lux | Non-responders to bevacizumab (Avastin) therapy of choroidal neovascular lesions[END_REF][START_REF] Cleary | Intravitreal bevacizumab in the treatment of neovascular age-related macular degeneration, 6-and 9-months results[END_REF][START_REF] Madhusudhana | Intravitreal bevacizumab (Avastin) for the treatment of choroidal neovascularization in age-related macular degeneration: results from 118 cases[END_REF]

Materials and Methods

The BEAT-AMD-Study is a prospective, double-blinded, randomised, monocentric, pilot-study, and was conducted at our clinic. The study and data accumulation were carried out with approval from the ethics committee of the city of Vienna and informed consent for research was obtained from all subjects.

Eight subjects with fibrovascular PED, subfoveal CNV extending under the geometric centre of the foveal avascular zone, and/or a macular thickness of at least 300µm in both eyes were included. Exclusion criteria were proteinuria or renal impairment, hepatic dysfunction, vision threatening ophthalmic diseases other than AMD, or a history of arterial thromboembolic diseases or cancer.

Examinations were performed before start of infusion therapy, on a weekly basis for the first 6 weeks, every second week for the following 6 weeks, then every 4 weeks for the next 12 weeks. The follow-up time was 24 weeks.

Measurement of BCVA, RVA, intraocular pressure (IOP), macular thickness, and complete slit lamp examination were performed at every visit, while fluorescein angiography (FA), and indocyanine green angiography (ICGA) were performed before infusion therapy and after 12 and 24 weeks. BCVA was determined using the ETDRS charts according to the guidelines of the Treatment of Age-related Macular Degeneration with Photodynamic Therapy (TAP) study. [START_REF]Tretament of Age-Related Macular Degeneration with Photodynamic Therapy (TAP) Study Group. Photodynamic therapy of subfoveal choriodal neovascularisation in age-related macular degeneration with verteporfin: oneyear results of 2 randomized clinical trials-TAP report[END_REF] It was performed at a test distance of 4 meters, with letter-by-letter scoring and a chart luminance of 100 candelas (cd)/m 2 . A loss or gain of up to 2 lines (10 letters) was considered as stable BCVA, a loss of 3 lines (15 letters) was considered as decrease in BCVA.

RVA was examined using a standardised, German-language reading test (Radner® reading charts). [START_REF] Radner | The validity and reliability of short German sentences for measuring reading speed[END_REF] The measurements were performed monocularly with the patient's optimal refractive correction at working-distance.

The macular thickness was measured using the Stratus OCT 3 (Carl Zeiss Meditec Inc.). Using the macular thickness program, 6 radial lines through the centre of the foveal avascular zone were performed. An internal fixation light was used to maintain the fixation throughout the examination, where possible.

FA was performed with the Heidelberg Retina Angiograph (Heidelberg Engineering GmbH, Heidelberg, Germany). 6-8 pictures were taken in the early phase after injection of fluorescein, then every 15 seconds, after 1 minute, 2, 5, and 10 minutes.

Distances in the angiograms were measured in mm with the help of the Heidelberger software. ICGA was also performed, including late frames after 20-30 minutes.

Subjects were looked after by an internal medical doctor (oncologist) throughout the entire study. Internal medical examinations including blood pressure, blood sampling, urine sampling, electrocardiogram, and weight measurement were performed throughout the first 6 weeks and after 24 weeks, in order to avoid any additional risk related to the administration of Avastin.

Subjects were randomised to receive either bevacizumab (Avastin®, Genentech Inc.) intravenous infusions, 3 times 5 mg/kg every 2 weeks (Avastin group) or sodium chloride 0.9% intravenous infusions, 3 times 100 ml every 2 weeks (NaCl group).

Randomisation was performed by the oncologist, by picking an envelope. As the present study is double-blinded, the oncologist and the pharmacist, who prepared the infusion, were the only ones informed about the treatment group the subject was randomised in to. The subject and the examining ophthalmologists were not informed about the randomisation result.

Data was collected and calculated using SPSS (Version 11.0). Statistical analysis was not performed due to the small number of subjects included. Data are presented Schmid-Kubista: BEAT-AMD-Study 4 as mean+SDM, percentage, and difference between measurements at different time points.

Results

Four subjects received Avastin systemic therapy and four subjects received NaCl infusions. 4 women and 4 men were included. Two women and two men received Avastin and the others received NaCl. The mean age was 79+4 (73-82 years) in the Avastin group and 74+9 (65-83 years) in the NaCl group. All eyes were phakic.

Before treatment the BCVA in the Avastin and in the NaCl group was 0.33+0.4 and 0.56+0.33 respectively, while the RVA was 0.6+0.5logRAD and 0.5+0.4logRAD respectively. At that time the macular thickness and the lesion size were 375.9+76.6µm and 5.42+2.15mm respectively in the Avastin group (Figures 1 and2) and 263.2+81.2µm and 3.72+1.88mm in the NaCl group (Figures 3 and4). The mean blood pressure was 140/80mmHg in both groups. The IOP was 16+1mmHg in the Avastin group and 16+4mmHg in the NaCl group.

At the end of follow up, after 24 weeks, the BCVA and RVA were 0.33+0.44 and 0.3+0.4logRAD respectively in the Avastin group and 0.53+0.42 and 0.5+0.4logRAD in the NaCl group. The macular thickness and lesion sizes were 272.3+91.5µm and 4.86+1.92mm respectively in the Avastin group (Figures 1 and2), 290.8+78.2µm and 3.93+1.93mm in the NaCl group (Figures 3 and4). The mean blood pressure remained 140/80mmHg in the NaCl group, while the systolic pressure increased to 150+12mmHg and the diastolic pressure increased to 90+12mmHg in the Avastin group. The IOP was 15+1mmHg in the Avastin group and 17+2mmHg in the NaCl group.

After 10 weeks the BCVA in the Avastin group dropped to 0.39+0.46, which was a difference of 0.07+0.06 compared to BCVA in the same group before start of therapy.

There were no differences in BCVA in the NaCl group and no differences in RVA in either group.

Throughout the 24 weeks the macular thickness decreased in the Avastin group by 103.6+14.9µm, while there was an increase of 22.1+8.4µm in the NaCl group Schmid-Kubista: BEAT-AMD-Study 5 (Table1). In the NaCl group the highest increase in macular thickness was found after 10 weeks, which was an increase of 133.7+20.4µm .In the Avastin group, the thinnest macular thickness was measured at 5 weeks, with a difference of 138+2.6µm. The lesion size decreased in the Avastin group from 6.1+2.2mm before treatment, to 5.1+1.9mm at week 12, and 5.6+2.1mm after 24 weeks. In the NaCl group the lesion size increased from 3.7+1.9mm prior to treatment, to 3.8+2.0mm after 12 weeks, and 3.9+1.9 at the end of follow up.

In both the Avastin and the NaCl group 5 eyes remained stable with the BCVA being +5 letters throughout the 24 weeks, 2 remained stable with +10 letters, and 1 showed a decrease with -15 letters.

Schmid-Kubista: BEAT-AMD- Study 6 In the FA all 8 eyes in the NaCl group showed to remain exudative, while in the Avastin group 4 (50%) eyes became quiet and fibrotic, 3 (37.5%) eyes remained exudative and 1 (12.5%) eye had a subretinal bleeding. The subretinal bleeding began in week 10, while the other eye turned fibrotic.

Discussion

When Michels and co-authors published the idea of systemic Avastin treatment against AMD, [START_REF] Michels | Systemic Bevacizumab (Avastin) therapy for neovascular age-related macular degeneration[END_REF] the idea of systemic anti-VEGF treatment against AMD was implicated. The idea of treating both eyes at the same time was also very tempting,

since it has been proven that patients with occult CNV with serous PED in the first eye have a significantly higher risk of visual loss in the second eye. [START_REF] Pauleikhoff | Visual prognosis of second eyes in patients with unilateral late exudative age-related macular degeneration[END_REF] A systemic treatment with initial 3 infusion therapies over 6 weeks seemed very promising.

Lesions too big for PDT had no treatment option, since intravitreal anti-VEGFs were not yet available and they were thought to be very invasive bearing the risk of endophthalmitis. Subretinal surgery[12-14] was too invasive for those eyes too.

The advantage of our BEAT-AMD-Study, compared to the SANA-Study, [START_REF] Michels | Systemic Bevacizumab (Avastin) therapy for neovascular age-related macular degeneration[END_REF] is that our study is randomised, double-blinded, and placebo controlled. Neither the patient, nor the investigating ophthalmologist knew which treatment arm the patient was randomised into. We began our study in August 2005 and recruited until January 2008. However, recruitment showed to become more difficult, after the idea of the off-label use of Avastin intravitreal injections has worked itself out. [START_REF] Rosenfeld | Optical coherence tomography findings after intravitreal injection of bevacizumab (Avastin) for neovascular age-related macular degeneration[END_REF][START_REF] Avery | Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration[END_REF] Those intravitreal injections of anti-VEGFs [START_REF] Study | Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration[END_REF][START_REF] Michels | Ranibizumab therapy for neovascular age-related macular degeneration[END_REF] showed not to be as invasive as assumed, [START_REF] Avery | Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration[END_REF] only few patients develop exudative AMD on both eyes, and of these most have some internal medical problem, which did not allow inclusion into the BEAT-AMD-Study. Due to those reasons we decided to discontinue recruitment and publish our data.

Our main outcome measure was lesion size and we were able to show a decrease of 0.5+0.08mm in the Avastin group throughout the 24 weeks. Macular thickness and visual acuity were additional outcome measures, whereby we found decreases in macular thickness in the Avastin group already within the first two weeks, with a difference of 100.7+4.5µm lasting until week 24. However, neither the BCVA, nor the RVA, showed improvement in this group. Unfortunately, there was a decrease in Schmid-Kubista: BEAT-AMD- Study 7 BCVA in week 10 by 0.07+0.06, which may be due to the development of the subretinal bleeding in one eye of a patient in week 10. Furthermore, there was a slight rise in blood pressure in this group, which began with 140/80mmHg and ended with the systolic pressure being 150+12mmHg and the diastolic pressure being 90+12mmHg. The rise in blood pressure was controlled and treated by the specialist for internal medicine and did not exceed 160/100mmHg in any patient.

In the placebo control group there was an increase in lesion size throughout the 24 weeks. BCVA and RVA remained stable. Although, after 24 weeks, macular thickness was comparative to the thickness before treatment, a big increase was found at week 10, which showed a difference of 133.7+20.4µm compared to prior treatment. Blood pressure remained stable.

When defining the BCVA by loss of lines, both groups had the same outcome. In both groups only one eye showed a loss of 3 lines (15 letters), while the other eyes remained stable. However, the FA in week 24 showed that 50% of the eyes turned quiet and fibrotic, 37.5% remained unchanged and 12.5% developed a subretinal bleeding in the Avastin group, while all 8 eyes remained exudative in the NaCl group.

IOP remained unchanged in both groups.

Even though the macular thickness got smaller in the Avastin group, the BCVA did not get better. In the NaCl group the BCVA and the macular thickness remained stable. This might be due to the fibrovascular reaction of the macula in the Avastin group, developing a fibrotic scar, with low BCVA, while a PED with a greater macular thickness still seems to have an equivalent BCVA.

Concluding we can say, that the lesion size and the macular thickness decreased in the Avastin group. The OCT and FA showed that the macula turns quiet and fibrotic

Table 1 :

 1 Macular thickness in both groups throughout the study. (Pre= before infusion

	therapy)
		Avastin Group	NaCl Group
		Macular Thickness (µm)	Macular Thickness (µm)
	Pre	375.9+76.6	263.2+81.2
	Week 1	306.9+144.8	295.8+99.6
	Week 2	275.2+81.1	292.3+119.8
	Week 3	271.9+94.7	282.3+96.9
	Week 4	303.4+164.0	326.5+39.5
	Week 5	237.9+79.2	367.0+39.6
	Week 6	259.1+116.2	339.3+95.0
	Week 8	258.3+116.7	374.0+75.9
	Week 10	251.3+81.9	400.8+44.3
	Week 12	279.4+145.9	314.8+104.4
	Week 16	235.2+69.0	333.8+89.4
	Week 20	239.9+58.9	339.8+46.6
	Week 24	272.3+91.5	290.8+78.2

  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 inBJO 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). Spaide RF, Sorenson J, Maranan L. Combined photodynamic therapy with verteprofin and intravitreal triamcinolone acetonide for choroidal neovascularization. Ophthalmology. 2003;110:1517-1525. 11 Rechtman E, Danis RP, Pratt LM, et al. Intravitreal triamcinolone with photodynamic therapy for subfoveal choroidal neovascularisation in age related macular degeneration. Br J Ophthalmol. 2004;88:344-347. 12 Binder S, Stolba U, Krebs I, et al. Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularisation resulting from agerelated macular degeneration: A pilot study. Am J Ophthalmol. 2002;133:215-Krebs I, Hilgers RD, et al. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest Ophthalmol Vis Sci. 2004;45:4151-4160. 14 Thomas MA, Grand MG, Williams DF, et al. Surgical management of subfoveal choriodal neovascularisation. Ophthalmology. 1992;99:952-968. 15 Kabbinavar FF, Schulz J, McCleod M, et al. Addition of Bevacizumab to Bolus Fluorouracil and Leucovorin in First-Line Metastatic Colorectal Cancer: Results of a Randomized Phase II Trial. J Clin Oncol. 2005;23:3697-3705.

		Schmid-Kubista: BEAT-AMD-Study 8
	refuse an intravitreal injection, and patients who have had previous vitrectomy should
	be considered for this treatment option.
	Licence for Publication
		225.
	13	Binder S,
	under Avastin systemic treatment. Development of BCVA and RVA are comparable
	in both groups and did not show any difference. Avastin has systemic side effects

such as arterial hypertension and thrombosis. Due to its systemic side effects, expensive costs and comparable results with intravitreal injections we suggest offering this systemic therapy to selected patients. After careful selection, according to thrombosis anamnesis, patients with exudative AMD in both eyes, patients who

Funding

There was financial support by the "Scientific Grant of the Mayor of Vienna", received in June 2006.

Competing Interest: None declared.

Appendices

Figures 1 and2: Displaying the FA and OCT from both eyes of one patient who received systemic treatment with Avastin. The FA and OCT pictures were taken before treatment (top), after 12 weeks (middle), and after 24 weeks (bottom).

Figures 3 and4: Displaying the FA and OCT from both eyes of one patient who received NaCl. The FA and OCT pictures were taken before infusion (top), after 12 weeks (middle), and after 24 weeks (bottom).