Marco Ciancaglini
email: m.ciancaglini@unich.it

Paolo Carpineto

Luca Agnifili

Mario Nubile

Vincenzo Fasanella

Peter A Mattei

Leonardo Mastropasqua

Conjunctival characteristics in primary open angle glaucoma and modifications induced by trabeculectomy with mitomycin C: an in vivo confocal microscopy study

Keywords: filtering blebs, trabeculectomy, confocal microscopy, conjunctival microcysts

Introduction

The basic mechanism of all filtering procedures is the creation of a fistula at the limbus which allows aqueous humor to drain from the anterior chamber, thereby circumventing pathologic obstruction to outflow. The aqueous thereby flows directly or indirectly into subconjunctival spaces and subsequently removed by one or more mechanisms. [START_REF] Benedikt | The effects of filtering operations[END_REF][START_REF] Teng | Histology and mechanism of filtering operations[END_REF] Trabeculectomy is the most effective filtering surgical procedure for lowering intraocular pressure. [START_REF] Jones | Recent advances in trabeculectomy technique[END_REF] The majority of successful trabeculectomy are characterized by an elevation of the conjunctiva at the surgical site, commonly referred to as a filtering bleb.

Currently, besides the standard clinical assessment, [START_REF] Picht | Classification of filtering in trabeculectomy: biomicroscopy and functionality[END_REF][START_REF] Cantor | Morphologic classification of filtering blebs after glaucoma filtration surgery: the Indiana Bleb Appearance Grading Scale[END_REF][START_REF] Migdal | The developing bleb: effect of topical prostaglandins on the outcome of glaucoma fistulising surgery[END_REF][START_REF] Addicks | Histologic characteristic of filtering blebs in glaucomatous eyes[END_REF] several morphological diagnostic methods such as laser scanning in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT), permit a precise analysis of the filtering bleb structure and function after glaucoma surgery by analyzing the conjunctival and sub-conjunctival features. [START_REF] Labbé | In vivo confocal microscopy study of blebs after filtering surgery[END_REF][START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF][START_REF] Guthoff | In Vivo Confocal Microscopy of Failing and Functioning Filtering Blebs: Results and Clinical Correlations[END_REF][START_REF] Ciancaglini | Filtering Bleb Functionality: a Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study[END_REF] Several studies have investigated the ability of IVCM in the microscopic evaluation of the bleb tissue, analyzing the drainage capability of the filtering blebs by assessing the presence of epithelial microcysts (defined as intraepithelial and extracellular optically empty spaces), the density of sub-epithelial connective tissue and the presence of blood vessels.

We recently reported the presence of epithelial microcysts within the conjunctiva either on eyes affected with untreated ocular hypertension or medically treated glaucoma eyes. [START_REF] Ciancaglini | Conjunctival Modifications in Ocular Hypertension and Primary Open Angle Glaucoma: An In Vivo Confocal Microscopy Study[END_REF] To date, to the best of our knowledge, the assessment and comparison before and after surgery of the features of conjunctiva at the site of trabeculectomy have not been reported. The aim of our study was to evaluate the microscopic epithelial features seen with IVCM of bulbar conjunctiva of glaucomatous patients undergoing trabeculectomy in order to elucidate modifications induced by surgery. Specifically, we compared the characteristics of the bulbar conjunctiva using IVCM in patients with glaucoma prior to and one month after trabeculectomy with an age and gender matched group of glaucoma patients who continued medical treatment.

Materials and methods

A comparative case series study was performed. The study adhered to the tenets of the Declaration of Helsinki and informed consent was obtained from all patients prior to their enrollment. We examined 15 eyes (8 right and 7 left eyes) of 15 consecutive Caucasian patients referred to the Ophthalmic Clinic of University Chieti-Pescara, Italy, 10 males and 5 females ; (mean (SD) age 64.5 (5.1) years; range 54-72) referred to the Ophthalmic Clinic of University Chieti-Pescara, Italy,affected with POAG and scheduled to undergo fornix based flap trabeculectomy, performed as previously described [START_REF] Jones | Recent advances in trabeculectomy technique[END_REF] .

Inclusion criteria were the followings: uncontrolled IOP (≥22 mmHg, mean of three measurements at 9 AM, 12 noon and 4 PM) under maximal tolerated medical therapy, topical hypotensive therapy started at least 12 months before the day of IVCM examination and unmodified during the last 2 months, progression of glaucomatous damage confirmed on three consecutive SITA 30-2 visual field, use of mitomycin C (MMC) 0.2 mg/mL for 2 min applied on the sclera at the site and posterior to the site where the flap was outlined. The results of surgery were classified as success when a reduction of one-third of preoperative IOP without antiglaucoma treatment was achieved. [START_REF] Edmunds | Factors associated with success in first-time trabeculectomy for patients at low risk of failure with chronic open-angle glaucoma[END_REF] Ten consecutive glaucomatous patients with open angle glaucoma but with an acceptable target pressure with medical therapy (6 males and 4 females; (mean (SD) age 63.3 (4.1) years) were used as controls.

Inclusion criteria were: IOP lower than 20 mmHg with medical therapy unmodified in the last 6 months and no history of progression on visual field examination (Humphrey 30-2 full-threshold) during the last 12 months.

All eyes enrolled in the study showed a best corrected visual acuity ≥8/10, a refractive error ≤4 diopters (spherical equivalent), and a corneal pachymetry ranging from 530 to 560 mμ. Overall exclusion criteria were: a history of ocular trauma, aphakia, previous trabeculoplasty or surgical procedure for glaucoma, uveitic glaucoma, contact lens wearing, the presence of any other ocular diseases in the last twelve months except open angle glaucoma, and end stage glaucoma.

Each glaucomatous eye scheduled to undergoing trabeculectomy and one eye randomly chosen in the control group, were carefully examined using digital confocal Laser-Scanning Microscope (LSM) (HRT II Rostok Cornea Module, Heidelberg Engineering GmbH, Germany) by a single operator masked for patient status (MN).

The technical characteristics of this instrument and the details of conjunctival examination were previously described. [START_REF] Mastropasqua | Corneal and conjunctival manifestations in Fabry Disease: in vivo Confocal Microscopy study[END_REF] The confocal assessment of bulbar conjunctiva was performed at baseline for all enrolled eyes (one week before trabeculectomy in eyes scheduled for surgical therapy).

Patients were seated in front of the microscope, with their head steadied with the aid of a headrest and with the eye properly aligned in order to obtain a tangential optical section of the superior bulbar conjunctiva.

They were asked to follow a dedicated mobile bright red light provided with the instrument with the fellow eye. A digital camera provided a lateral view of the eye and objective lens in order to check the position of the objective lens on the surface of the eye for each scan. LSM objective was positioned gently in contact with the ocular surface separated by a PMMA (polymethyl methacrylate) contact cap and a drop of 0.2% polyacrylic gel (Viscotirs® Gel, CIBA Vision® Ophthalmics, Marcon, Venezia, Italy) served as coupling medium. Sequential images 300X300 mμ in size derived from automatic scans and manual frames were acquired of the upper bulbar conjunctiva at the intermediate layer (10 to 20µm) of each examined eye, 2 mm from the limbus at the site of the planned trabeculectomy, in downward gaze. Results were reported as the average of six images selected by the IVCM operator (MN) in a masked fashion from about 40 images.

For the IVCM assessment, we microscopically evaluated the bulbar conjunctiva on identifying epithelial microcysts, such as those reported in the conjunctival wall of functioning filtering blebs. [START_REF] Labbé | In vivo confocal microscopy study of blebs after filtering surgery[END_REF][START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF][START_REF] Guthoff | In Vivo Confocal Microscopy of Failing and Functioning Filtering Blebs: Results and Clinical Correlations[END_REF][START_REF] Ciancaglini | Filtering Bleb Functionality: a Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study[END_REF] The main parameters considered in the IVCM examination were the mean microcyst density (cysts/mm 2 ) and the mean microcyst area (µm 2 ). The surface area of epithelial microcysts was calculated using ImageJ, an open source software (http://rsb.info.nih.gov/ij), as described elsewhere. [START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF] Additionally, in order to verify the findings obtained in the sampled fields, we extended the analysis area to the whole conjunctival superior region (1.2 X 3 mm, approximately) in 3 eyes undergoing trabeculectomy and 3 control eyes. A planar reconstruction was thus obtained permitting the evaluation of the distribution of epithelial microcysts. Final maps were obtained by juxtapositional acquisition of about 100 images by moving the cornea module from the nasal to temporal region (right and left side of images, respectively) and from the limbus to the upper lid (bottom and top of images, respectively).

The IVCM procedure was repeated in all eyes 6 weeks later.

The eyes that underwent surgical therapy initiated unpreserved topical steroids for 4 weeks (unpreserved dexamethasone 0.15% eye drops four times a day for 2 weeks and three-time daily for the following 2 weeks) and topical antibiotic for the initial 2 weeks (ofloxacin 0.3% fourfold daily). IVCM was repeated 5 weeks after trabeculectomy, that is one week after cessation of any topical therapy. During the study period, control eyes continued previous topical antiglaucoma therapy.

Statistical analysis was performed using SPSS ® Advanced Statistical TM 13.0 Software (2005, Chicago, IL, USA). Differences in trabeculectomy and control groups at baseline were evaluated using Student's t test 6 and chi-squared, where appropriate. McNemar test was performed to compare differences of mean microcyst density and area at baseline and at the end of the study in both groups. Data prior to transformation are reported and a logarithmic transformation of mean microcyst density and area values was applied prior to statistical analysis in order to correct the wide sample data distribution.

Results

All the data are presented as mean±STD. The demographic and clinical data of each group at baseline are shown in Table 1. Significant differences for age and sex were not observed. In the eyes scheduled to undergo trabeculectomy the mean pre-operative IOP was 25.1±3.2 mmHg with a median of 3 daily drugs per eye. Antiglaucomatous medication included betablockers (n=15), prostaglandins (n=15), brimonidine (n=12) and carbonic anhydrase inhibitors (n=8), in various combination. At baseline, conjunctiva frequently showed irregular epithelial features, characterized by large and polygonal cells with low uniformity in size and shape and hyper reflective borders. Epithelial microcysts were reported in all cases as structures optically clear, occasionally with features of round cells (presumably representing inflammatory cells) and amorphous material (probably representing proteinaceous debris) [START_REF] Shields | Clinical and histopathologic observations concerning hypotony after trabeculectomy with adjunctive mitomycin C[END_REF] partially filling the inner space, often surrounded by a well defined hyper-reflective and mildly thick wall (capsule), round or oval-shaped and differently sized. When observing the planar reconstruction of the superior bulbar conjunctiva compounded at baseline examination, we found a relatively homogeneous distribution pattern of microcysts with slight tendency towards clustering (Figure 1). With regards to the main parameters analyzed, we documented a mean microcyst density of 22.4±11.9 (cysts/mm 2 ) and a mean microcyst area of 4696.0±3608.1 (µm 2 ), expression of an evident and high inter-individual variability.

No significant intra-and post-operative complications occurred, except for 2 cases of ocular hypotony and athalamia, resolved within 3 days without surgical treatment. In the post-operative period no eyes received 5-fluororacyle injections and topical therapy (antibiotic/steroid fixed combination) was discontinued after 4 weeks.

Five weeks after trabeculectomy and then 6 weeks after baseline the mean IOP was 16.1±1.7 mmHg without medical therapy and all the eyes were classified as success, that is with a reduction of one-third of preoperative IOP (without medical therapy).

Clinical evaluation of the trabeculectomized eyes (Moorfields Bleb Grading System (MBGS) criteria) [START_REF] Wells | Comparison of Two Clinical Bleb Grading Systems[END_REF] indicated a diffuse bleb pattern in all cases. The IVCM examination of conjunctiva showed differentshaped and sized microcysts, larger and less uniformly disposed than in the pre-surgical epithelium, surrounded by a low reflective and thinner wall, frequently with features of cohesion realizing a multicluster distribution, more evident at the bleb site (Figure 2). Also at this level, the inner space of the epithelial microcysts showed modifications, occasionally appearing filled with amorphous material and/or round cells and sometimes also with amorphous material as found in the preoperative examination.

When analyzing the microcysts characteristics, a mean density of 103.1±22.6 (cysts/mm 2 ) and mean area of 29489.3±12954.9 (µm 2 ) where observed. These parameters were statistically different and approximately five time greater with respect to preoperative values (p=0.003).

In the control group, mean IOP was 15.7±1.9 mmHg, with a median of 2 daily drugs per eye, values statistically different (p<0.001) with respect to eyes undergoing trabeculectomy. Antiglaucomatous medication included betablockers (n=9), prostaglandins (n=7), brimonidine (n=2) and carbonic anhydrase inhibitors (n=1), in various combination.

At baseline analysis, both epithelium or microcysts distribution and features appeared with the same characteristics described in patients undergoing filtering surgery, without evident differences (Figure 3).

When evaluating the microcysts parameters, the mean density and mean area values were 26.3.±10.6 (cysts/mm 2 ) and 5797.0±4021.0 (µm 2 ), respectively, which were not significantly different from those reported in patients before trabeculectomy. At 6 weeks, the mean IOP was 15.9.±1.7 mmHg, the epithelial and microcysts features were substantially unmodified with respect to baseline (Figure 4). Also, the microcysts parameters did not show statistically significant modifications: mean microcyst density and mean microcyst area values of 26.5±11.8 (cysts/mm 2 ) and 6096.9±3389.1 (µm 2 ), respectively.

In both groups of patients we did not observed signs of inflammatory dendritic or round cell infiltration within the epithelium.

Discussion

The main challenge of filtering surgery is to induce a decrease of pre-operative IOP by increasing aqueous outflow via a new pathway, thereby reducing the progression of glaucoma. Among diagnostic methods available in the clinical practice, the slit-lamp evaluation of the site of trabeculectomy yields important information concerning the conjunctival bleb characteristics such as extension, elevation and vascularization. Currently, this method is considered a reliable procedure to clinically assess tissue modifications induced by trabeculectomy. Recently, the use of IVCM provided additional information in filtering bleb examination because it permitted a microscopic analysis of specific conjunctival features such as the presence of epithelial microcysts and the density of sub-epithelial connective tissue. [START_REF] Labbé | In vivo confocal microscopy study of blebs after filtering surgery[END_REF][START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF][START_REF] Guthoff | In Vivo Confocal Microscopy of Failing and Functioning Filtering Blebs: Results and Clinical Correlations[END_REF][START_REF] Ciancaglini | Filtering Bleb Functionality: a Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study[END_REF] However, to date, these characteristics have been considered as only a sign of tissue rearrangement or modification, exclusively induced by the surgical procedure.

The results of this study showed that conjunctival modifications were pre-existant to trabeculectomy: epithelial microcysts appeared widely present and diffuse also in glaucomatous eyes under medical therapy before surgery. Therefore, surgery influenced a preexisting phenomenon in that either the eyes scheduled for trabeculectomy or control eyes under medical therapy showed similar features with regards to characteristics and distribution of microcysts, even though the IOP was quite different between the two groups and significantly higher in eyes undergoing surgical therapy. These observations are in agreement with those reported in an our previous study in which we demonstrated the absence of epithelial microcysts in healthy eyes and conversely, documented their presence in all eyes with increased IOP, affected with either untreated ocular hypertension or treated open angle glaucoma. [START_REF] Ciancaglini | Conjunctival Modifications in Ocular Hypertension and Primary Open Angle Glaucoma: An In Vivo Confocal Microscopy Study[END_REF] Furthermore, as confirmed by the repeated analysis of conjunctival epithelium in control group after 6 weeks, the presence and features of microcysts in glaucomatous eyes did not show significant modifications, at least not after the short period of time evaluated in this study.

The final effect of the filtering procedure was a five-fold increase of conjunctival microcysts density and surface area on the site of bleb with respect to preoperative values: a sign of a marked aqueous filtration enhancement through the sclera and, subsequently, the conjunctiva. A possible theory to explain these results is that an activation of transcleral outflow might be induced in medically treated glaucomatous eyes by the hyperbaric condition as an alternative pathway of humour aqueous drainage. But the presence of epithelial microcysts should not be considered a side effect of antiglaucoma drugs or preservatives, since these structures were also found in cases of untreated ocular hypertension. [START_REF] Ciancaglini | Conjunctival Modifications in Ocular Hypertension and Primary Open Angle Glaucoma: An In Vivo Confocal Microscopy Study[END_REF] However this adaptation mechanism by itself was not sufficient to re-establish a controlled IOP because, as previously reported, [START_REF] Jackson | Human Scleral Hydraulic Conductivity: Age-RelatedChanges, Topographical Variation, and Potential Scleral Outflow Facility[END_REF][START_REF] Toris | Aqueous humor dynamics in the ageing human eyes[END_REF] the potential in vitro human transcleral outflow capacity was 0.33 μl x min x mmHg in the suprachoroidal space: a much higher value than in vivo (1.1-1.5 μl/min). Base on these findings, filtering surgery is successful when transcleral outflow capability is substantially increased. The active role of the transcleral and trans-conjunctival aqueous percolation for the maintenance of bleb functionality in trabeculectomized eyes has been previously supposed 15,19,-20 and confirmed by several observations which suggested that a successful trabeculectomy yielding a strong decrease of postoperative IOP required the presence of low density subconjunctival connectival tissue and a high density and wide surface area of intra-epithelial microcysts. [START_REF] Labbé | In vivo confocal microscopy study of blebs after filtering surgery[END_REF][START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF][START_REF] Guthoff | In Vivo Confocal Microscopy of Failing and Functioning Filtering Blebs: Results and Clinical Correlations[END_REF][START_REF] Ciancaglini | Filtering Bleb Functionality: a Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study[END_REF] However two aspects of our study require further elucidation: the role of topical therapy in the pathogenesis of pre-operative conjunctival modifications and the effect of both intra-operative MMC and post-operative medications on post-surgical findings. To date, as previously mentioned, clear evidence that topical antiglaucoma drugs or preservatives are involved in the microcysts formation and persistence has not been reported. [START_REF] Ciancaglini | Conjunctival Modifications in Ocular Hypertension and Primary Open Angle Glaucoma: An In Vivo Confocal Microscopy Study[END_REF] In order to define the real influence of antiglaucoma drugs on reported conjuncival features, a prospective study evaluating the conjunctiva of native glaucomatous eyes before and after medical therapy is mandatory. We propose that MMC and steroids could have only a limited role in the development of the conjunctival modifications seen in our study, probably leading, with greater frequency, to higher microcyst diameters, to thinning of the microcysts wall, and to clustering and reduction of postoperative scarring. In accordance with our hypothesis, Labbè et al [START_REF] Labbé | In vivo confocal microscopy study of blebs after filtering surgery[END_REF] reported that epithelial microcysts were smaller in diameter in trabeculectomy without MMC (from 10 to150 µm) with respect to filtering surgery with MMC application (from 10 to 300 µm).

Furthermore, in the same study, the authors did find the presence of hyper-reflective microdots in the superficial epithelium layer (probably representing necrotic epithelial cells or inflammatory cells) only in eyes who received MMC and in no eye who did not.

Conversely, in an our previous study [START_REF] Ciancaglini | Filtering Bleb Functionality: a Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study[END_REF] in which we analyzed different epithelial parameters such as the microcysts density and surface of filtering blebs with and without the intra-operative use of MMC, we did not report any microscopic difference between patients who received intra-operative MMC and patients who did not. When analyzing sub-epithelial layers, Messmer et al [START_REF] Messmer | In vivo confocal microscopy of filtering blebs after trabeculectomy[END_REF] reported a bleb stroma organized with a less rarified collagenlike network and with less large cystic spaces in patients who did not receive intraoperative MMC with respect to patients who did. By considering all these findings, one may suggest that the use of intra-operative MMC affects the feature of the bleb stroma and the size of epithelial microcysts, but probably do not modify the total surface of intra-epithelial microscysts.

Recently Amar and coll. [START_REF] Amar | Filtering Blebs and Aqueous Pathway. An Immunocytological and In Vivo Confocal Microscopy[END_REF] using both impression cytology and in vivo confocal microscopy hypothesized that microcysts observed at the surface of functioning blebs seemed to correspond to goblet cells, mostly containing aqueous humor instead of highly hydrophilic gel-forming mucins. Therefore they suggested that the transcellular pathway of the aqueous humor could occur at the level of goblet cells toward the ocular surface. The authors proposed that larger microcysts observed with in vivo confocal microscopy could correspond to a confluence of adjacent empty or degenerating goblet cells, resulting in large clear spaces.

In our study the presence of conjunctival globet cells was not methodically investigated because we searched for anatomical modifications of so called conjunctival epithelial cysts induced by surgery and not for biological analysis of cell morphology or function. However, the hypothesis of biological modification of globet cells is an interesting features that deserves further investigation.

In conclusion, the analysis of conjunctiva in medically and surgically treated glaucomatous patients before and after trabeculectomy, provided additional and valuable information which clarified important physiopathological aspects of the aqueous outflow and the effects induced by filtering procedures.

Tables

Tables

Figure 1 .

 1 Figure 1. Planar reconstruction of the superior bulbar conjunctiva in a glaucomatous eye

Figure 2 .

 2 Figure 2. Planar reconstruction of the superior conjunctiva in a trabeculectomized eye. The

Figure 3 .

 3 Figure 3. Planar reconstruction of the superior bulbar conjunctiva in a glaucomatous eye

Figure 4 .

 4 Figure 4. Planar reconstruction of the superior bulbar conjunctiva in a glaucomatous eye

Table 1 : Characteristics of the Patients at baseline

 1

		Trabeculectomy	Controls
	Number of eyes	15	10
	Mean age (years)	64.5 ± 5.14	63.3 ± 4.11	P=ns*
	Gender (M/F)	10/5	6/4	P=ns**
	Mean IOP (mmHg)	25.1.±3.21	15.7±1.85	P=<0.001**
	Number of drugs	3.2±0.56	2.0±0.81	P=<0.001***
	MD	6.34±2.45	9.47±3.12	P<0.001**
	M = male; F = female; IOP= Intra Ocular Pressure; MD = mean defect
	* t test for indipendent samples
	** Chi square test

*** Mann-Whitney test

Table 1 : Characteristics of the Patients at baseline

 1

		Trabeculectomy	Controls
	Number of eyes	15	10
	Mean age (years)	64.5 ± 5.14	63.3 ± 4.11	P=ns*
	Gender (M/F)	10/5	6/4	P=ns**
	Mean IOP (mmHg)		15.7±1.85	P=<0.001**
	Number of drugs	3.2±0.56	2.0±0.81	P=<0.001***
	MD	6.34±2.45	9.47±3.12	P<0.001**
	M = male; F = female; IOP= Intra Ocular Pressure; MD = mean defect
	* t test for indipendent samples
	** Chi square test

*** Mann-Whitney test


Dr Wai Siene Ng

Pankaj K Mbchb

Agarwal

Sikander Mrcophth

Sidiki

Lorna Frcophth

Mckay

John Bscorthoptics

Townend

PhD Augusto Azuara-Blanco

The Effect of Socioeconomic Deprivation on Severity of Glaucoma at Presentation

Keywords: Socioeconomic status, Low-income population, Glaucoma, Blindness

Objective: To evaluate the influence of socio-economic factors on severity of glaucoma at presentation Methods: All newly diagnosed glaucoma patients at the University Hospitals-NHS, Aberdeen, and South Glasgow University Hospitals-NHS, in 2006, were included. Glaucoma was severe at presentation if there was a repeatable visual field loss with a Mean Deviation index greater than 12 dB in the Humphreys visual fields test or an absolute paracentral scotoma within the central 5 degrees of the visual fields. Home address was used to determine the Scottish Index of Multiple Deprivation (SIMD) rank. The SIMD rank, demographics and severity of glaucoma at presentation was investigated using general linear modelling.

Results: There were 48 patients with severe glaucoma and 74 patients with non-severe glaucoma. In four the severity could not be determined. Severity of glaucoma at presentation was significantly associated with SIMD rank, being most severe in patients from areas with the lowest ranks (p=0.026).

Age was a significant factor (p=0.024) with severe glaucoma being more common in elderly patients.

Conclusions: Age and socio-economic deprivation were associated with severity of glaucoma at presentation, with patients from areas of higher socio-economic deprivation presenting with more advanced glaucoma.

Introduction

In the developed world, glaucoma treatment is widely available and yet glaucoma is one of the leading causes of blindness. [START_REF] Quigley | The number of people with glaucoma worldwide in 2010 and 2020[END_REF] In the UK, glaucoma is the second most common cause of visual impairment after age related macular degeneration. [START_REF] Bunce | Leading causes of certification for blindness and partial sight in England & Wales[END_REF] As we approach an expanding aging population in the UK it will undoubtedly pose greater challenges to the national health service. It is well recognised that severity of glaucoma at presentation is a major factor in the development of glaucoma blindness. [START_REF] Chen | Blindness in patients with treated open-angle glaucoma[END_REF] Due to the asymptomatic nature of the disease, early diagnosis followed by treatment initiation is essential to prevent blindness from advanced glaucoma.

The adverse impact of socioeconomic deprivation on health and mortality is well recognised and Scotland's poorer health indicators within the UK have been attributed to higher levels of deprivation. [START_REF] Smith | Socioeconomic differentials in mortality: evidence from Glasgow graveyards[END_REF][START_REF] Carstairs | Deprivation: explaining differences in mortality between Scotland and England and Wales[END_REF] With regards to visual disorders, socioeconomic deprivation has been linked with late presentation of glaucoma in England in a case-control study performed 10 years ago. [START_REF] Fraser | Deprivation and late presentation of glaucoma: case-control study[END_REF] Severe amblyopia has also been associated with poor socioeconomic background. [START_REF] Smith | Social deprivation and age of presentation of amblyopic[END_REF] The goal of this study was to evaluate the effect of socioeconomic deprivation on severity of glaucoma at presentation in newly diagnosed glaucoma patients. Newly diagnosed patients were retrospectively identified and case notes were reviewed by two experienced clinicians for presence of glaucomatous damage in both eyes. All patients with glaucoma presenting to the two units were included in the study. Patient age and gender, highest recorded intraocular pressure (IOP), blindness, other ocular morbidity, visual field tests (each subject had at least two visual fields), and optic disc findings were noted. Blind patients did not have visual fields performed.

Methods

Diagnosis of glaucoma was based on optic disc and visual field findings [START_REF] Foster | The definition and classification of glaucoma in prevalence surveys[END_REF]. The criteria was reproducible visual field defects at presentation characteristic of glaucomatous damage (i.e. abnormal glaucoma hemi-field test, or two or more contiguous points on the pattern standard deviation (PSD) plot with P < 0.01 loss or greater, or three or more contiguous points with P < 0.05 loss or greater, or a 10-dB difference across the nasal horizontal midline at two or more adjacent points in the total deviation plot) in reliable tests and corresponding glaucomatous optic disc damage (absence or thinning of the rim, notching and cup-to-disc ratio asymmetry of more than 0.2 in similarly sized discs and eyes). Reliability of visual fields was based on a false positive or negative of less than 25%.

The severity of glaucoma in both eyes was recorded but only one eye was chosen for analysis (see below). The presence of severe glaucoma was pre-defined before data collection as a visual field loss of either (1) mean deviation (MD) index > 12 dB or (2) absolute paracentral scotoma within the central 5 degrees of visual fields. If visual fields were unreliable, presence of severe disc damage (cup-to-disc ratio of more than or equal to 0.9) was used to determine severe glaucoma. Visual field testing was done with Humphrey SITA fast 24-2 (Humphrey Instruments, San Leandro, California).

Patients with ocular hypertension (i.e. with no glaucomatous damage) or glaucoma suspects, those with secondary glaucoma, other co-morbidity such as cataract, age-related macular degeneration, or history of previous intraocular surgery (other than uncomplicated cataract surgery) were excluded.

The SIMD 2006 was used to assess socioeconomic status. It records seven domains (Current Income, Employment, Health, Education Skills and Training, Geographic Access to Services, including public transport travel times for the first time, Housing and Crime Domain). The SIMD data is presented at data zone level. The postcode of each patient was allocated to an SIMD rank. This was obtained by matching them to their data zones, taken from the website www.sns.gov.uk which correspond them to their individual ranks from the SIMD 2006 website (www.scotland.gov.uk/SIMD2006Data). [START_REF]The Scottish Index of Mean Deprivation[END_REF] The SIMD rank ranges from 1 to 6505, 1 being the most deprived.

One eye per patient was used for analysis as the unit of analysis was the patient. The worst eye (according to severity of glaucoma) was chosen for statistical analysis. If both eyes had severe disease, the more severe one was included. Statistical analysis was performed by a senior statistician (John A Townend).

The effect of SIMD rank on the number of patients who presented with severe glaucoma was investigated using binary logistic regression. We also investigated the effect of SIMD rank on visual field MDs by using linear regression. MD values were square root transformed (using the absolute value of MD) prior to analysis to meet the requirement of normally distributed residuals. Regression analyses were adjusted for patient age and the effects of highest intraocular pressure and area (Aberdeen and Glasgow) were tested as possible confounding factors using forward selection.

Analyses were carried out using SPSS Ver 15.0 (SPSS Inc, Chicago). P-values < 0.05 were taken as statistically significant. The study was approved by both local ethics committees.

Results

In the period under study, 126 patients, 64 (50.8%) females and 62 (49.2%) males were included in the study. The mean age of patients was 71.6 (S.D = 12.5) years. There were 48 patients diagnosed as severe glaucoma, 74 as non-severe glaucoma and four in whom severity could not be determined due to absence of reliable visual field data. In these four patients, one had severe glaucomatous optic disc damage. Thirteen patients had PACG and 113 had POAG. Seventy patients had bilateral glaucoma and eleven had severe glaucoma. Two patients were registered blind due to glaucoma at the time of diagnosis. Table 1 illustrates SIMD ranks and number of patients with severe glaucoma.

The number of patients presenting with severe glaucoma was significantly associated with SIMD rank, being most common in patients from areas with the lowest ranks (most deprived areas) (p = 0.026). Age was also a significant factor (p = 0.024) with severe glaucoma being more common in older patients. Highest intraocular pressure and city of residence were not significant factors.

Increasing age was significantly associated with more negative visual field MD values (p =0.001). To evaluate whether more elderly patients were living in areas of low ranking SIMD, the analyses was stratified and found that the effect of age independently played a part in determining whether a person is likely to have severe glaucoma at presentation. SIMD rank, IOP and city of residence showed no significant association with MD scores. with ten, four and four respectively. [START_REF] Jarman | Identification of underprivileged areas[END_REF][START_REF] Townsend | Health and deprivation. Inequality and the north[END_REF][START_REF] Carstairs | Deprivation and health in Scotland[END_REF] The SIMD data is presented at data zone level with a median population size of 769, enabling small pockets of deprivation to be identified. Hence, this results in a comprehensive picture of relative area deprivation across Scotland. The SIMD has been extensively used in studies on mortality, cardiovascular disease as well as patient literacy. [START_REF] Howieson | Multiple deprivation and excess winter deaths in Scotland[END_REF][START_REF] Dani | Audit of literacy of medical patients in north Glasgow[END_REF][START_REF] Woodward | SIGN group on risk estimation. Adding social deprivation and family history to cardiovascular risk assessment: the ASSIGN score from the Scottish Heart Health Extended Cohort (SHHEC)[END_REF] Our study suggests patients who reside within areas of socioeconomic deprivation presented more frequently with severe glaucoma. This agrees with a previous case-control study done in England a decade ago, which found an association between lower socioeconomic status and late presentation with glaucoma. [START_REF] Fraser | Deprivation and late presentation of glaucoma: case-control study[END_REF][START_REF] Fraser | Risk Factors for Late Presentation in Chronic Glaucoma[END_REF] Poverty has also been linked to blindness in a Pakistan study. [START_REF] Gilbert | Poverty and blindness in Pakistan: results from the Pakistan national blindness and visual impairment survey[END_REF] Lower socioeconomic status has been linked to late presentation in several diseases such as breast, colorectal and skin cancer. [START_REF] Wells | Stage at diagnosis in breast cancer: race and socioeconomic factors[END_REF][START_REF] Powe | Fatalism amongst elderly African-Americans. Effects on colorectal cancer screening[END_REF][START_REF] Robinson | Socioeconomic status and attitudes of 51 patients with basal and squamous cell carcinoma and paired controls[END_REF] Presentation with advanced glaucoma has been associated with a poor visual prognosis and blindness. [START_REF] Grant | Why do some people go blind from glaucoma?[END_REF][START_REF] Klein | Prevalence of Glaucoma[END_REF][START_REF] Mikelburg | The rate of progression of scotomas in glaucoma[END_REF] Our finding highlights the need to recognise socioeconomic deprivation as a risk factor for late presentation of glaucoma and probable increased risk of blindness. [START_REF] Chen | Blindness in patients with treated open-angle glaucoma[END_REF] Upon diagnosis response to treatment depends on patient compliance, requiring education on glaucoma and its treatment. As the SIMD includes educational skills, it may be relevant that low literacy have been associated with low compliance to glaucoma therapy. [START_REF] Muir | Health literacy and adherence to glaucoma therapy[END_REF] Unsurprisingly, there was a statistically significant association between severity of glaucoma at presentation and the elderly as POAG is known to be an age-related disease. [START_REF] Friedman | Prevalence of open-angle glaucoma among adults in the United States[END_REF] It is possible that these elderly patients were not regular attendees at their optometrists as suggested by the General Household Survey which showed a reduction in (free) sight testing in those aged more than 65 years in the United Kingdom. [START_REF] Household | Analysis of Ophthalmic Data 1990-91 to 1993-94[END_REF] On the other hand, the possibility of having suboptimal detection services in the community also arises. Currently in the United Kingdom, the optometrist plays a major role in glaucoma detection.

Table 1 Scottish Index of Mean Deprivation Ranks and number of patients

Scottish Index of Mean Deprivation Ranks

In addition, not only are the deprived and elderly more likely to present late with severe glaucoma, but evidence suggests that they are more likely to lack awareness for the benefits of blind registration and support. [START_REF] Williams | Quality of life and visual rehabilitation: an observational study of low vision in three general practices in West Glamorgan[END_REF] This inevitably compounds onto their disability and serves to emphasize the importance of identifying such patients.

To date, literature linking severity of glaucoma and socioeconomic deprivation is scarce. [START_REF] Fraser | Risk Factors for Late Presentation in Chronic Glaucoma[END_REF] They also found also a strong association between IOP and advanced visual field loss at presentation but age was not a statistically significant factor. Our study offers an up-to-date report with a more complete assessment of deprivation using the SIMD for which we are the first to apply to glaucoma patients.

Screening of the UK adult population for glaucoma does not appear to be cost-effective from the point of view of the NHS although in Finland a recent cost-effectiveness analysis suggested that screening could be cost-effective. [START_REF] Burr | The clinical effectiveness and cost-effectiveness of screening for open angle glaucoma: a systematic review and economic evaluation[END_REF][START_REF] Vaahtoranta-Lehtonen | Cost effectiveness and cost utility of an organized screening programme for glaucoma[END_REF] Lower socioeconomic status is associated with lower uptake of screening programmes such as mammography and cervical screening. [START_REF] Calle | Demographic predictors of mammography and Pap smear screening in US women[END_REF] However, in light of our findings, targeted surveillance at high risk patients such as in areas of high deprivation and the elderly might need to be considered to help in early identification, preventing severe glaucoma and eventual disabling blindness.

Competing Interests: nil Funding: nil

Acknowledgments: nil

The Corresponding Author, Dr Wai Siene Ng 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 licencees, to permit this article (if accepted) to be published in BJO and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence (http://bjo.bmjjournals.com//ifora/licence.pdf)

A

  one-year hospital-based cross-sectional study was performed on all newly diagnosed patients with glaucoma (either primary open angle glaucoma[POAG], or primary angle-closure glaucoma, [PACG]) seen at the Aberdeen Royal Infirmary and the South Glasgow University Hospitals, NHS Trusts, Glasgow from the 1 st of January 2006 to the 31 st of December 2006.

Discussion

  Several scores have been utilized to classify socioeconomic deprivation, including Carstairs, Jarman, Townsend and the Scottish Index of Multiple Deprivation (SIMD). The SIMD records seven domains (Current Income, Employment, Health, Education Skills and Training, Geographic Access to Services, including public transport travel times for the first time, Housing and Crime Domain) of which there are 37 indicators of deprivation which allows for a more complete assessment of deprivation. On the other hand, Jarman, Townsend and Carstairs have fewer indicators of deprivation

  Fraser et al. have conducted a case control study in 1996 to 1997 to identify risk factors for advanced glaucoma as well as socioeconomic risk factors and late presentation of glaucoma. [6,16] They used Jarman's underprivileged area score to classify deprivation which only uses three factors (occupational class, housing tenure and access to a car) to measure individual deprivation. Fraser et al. reported a strong association between socio-economic deprivation and late presentation of glaucoma.


MD Christoph M E Deuter
email: christoph.deuter@med.uni-tuebingen.de

Ina Kötter

Ilhan Günaydin

Nicole Stübiger

Deshka G Doycheva

Manfred Zierhut

EFFICACY AND TOLERABILITY OF INTERFERON ALPHA TREATMENT IN PATIENTS WITH CHRONIC CYSTOID MACULAR EDEMA DUE TO NON-INFECTIOUS UVEITIS

Keywords: Uveitis, Cystoid Macular Edema, Treatment, Interferon Alpha

Aim: To assess the efficacy and tolerability of interferon (IFN) alpha in chronic cystoid macular edema (CME) due to non-infectious uveitis.

Methods: Retrospective analysis of an interventional case series. IFN alpha-2a was administered at an initial dose of 3 or 6 million IU per day subcutaneously and tapered afterwards to the lowest possible dose to maintain the absence of CME. Treatment efficacy was assessed by optical coherence tomography. Results: Twenty-four patients with chronic CME (median duration 36.0 months) due to non-infectious anterior (n=2), intermediate (n=18) or posterior (n=4) uveitis have been analysed. Ineffective pre-treatment included systemic corticosteroids (all patients), acetazolamide (22 patients) and at least one immunosuppressive drug (18 patients). IFN therapy was shown to be effective (= complete resolution of CME within 3 months, able to taper IFN) in 15 patients (62.5%), partly effective (= incomplete resolution of CME, unable to taper IFN) in 6 patients (25.0%) and not effective (= no response or recurrence of CME) in 3 patients (12.5%). IFN treatment was generally well tolerated. Common side effects including flu-like symptoms, fatigue or increased liver enzymes were dose dependent and led to discontinuation of IFN in only 1 patient. Conclusion: Our data demonstrate IFN alpha to be an effective and well-tolerated therapy for chronic refractory uveitic CME.

Introduction

Cystoid macular edema (CME) is a serious complication of uveitis affecting approximately one-third of patients. [START_REF] Lardenoye | Impact of macular edema on visual acuity in uveitis[END_REF] It represents one of the major causes of legal blindness in patients with intraocular inflammation. [START_REF] Okhravi | Cystoid macular edema in uveitis[END_REF] Therefore, CME legitimates aggressive medical treatment including systemic or intravitreal corticosteroids, acetazolamide and immunosuppressive drugs. [START_REF] Rothova | Medical treatment of cystoid macular edema[END_REF][START_REF] Zierhut | Treatment of uveitic macular edema with acetazolamide[END_REF] When a CME occurs in a condition of active uveitis, its management is mostly unproblematic if an early and adequate treatment of the intraocular inflammation is initiated.

In contrast, chronic uveitic CME still remains a therapeutic challenge. For us a CME is defined as 'chronic' if the uveitis itself appears quiescent (either burned out or well controlled by drugs) but, nevertheless, a CME is present that no longer responds to conventional anti-inflammatory therapy. For this refractory sight-threatening condition new therapeutic approaches need to be evaluated. Interferon (IFN) alpha is a cytokine belonging to the subgroup of type I interferons that exerts strong antiviral, antiproliferative and various immunomodulatory effects. It is approved for the treatment of viral hepatitis and myeloproliferative syndromes, as well as for certain solid tumours and lymphomas. [START_REF] Koetter | Use of interferon-α in Behçet´s disease[END_REF] However, during recent years IFN alpha has also been shown to be a very effective treatment for patients suffering from Behçet´s disease (BD) with inflammatory involvement of the posterior eye segment [START_REF] Koetter | The use of interferon α in Behçet disease: Review of the literature[END_REF][START_REF] Deuter | Behçet´s disease: ocular effects and treatment[END_REF] as well as for several cases of otherwise refractory uveitis. [START_REF] Bodaghi | Efficacy of interferon alpha in the treatment of refractory and sight threatening uveitis: a retrospective monocentric study of 45 patients[END_REF][START_REF] Plskova | Interferon-alpha as an effective treatment for noninfectious posterior uveitis and panuveitis[END_REF] A particular finding in the BD patients was that IFN alpha treatment without additional acetazolamide or immunosuppressives led to complete resolution of CME. [START_REF] Koetter | Human recombinant interferon alfa-2a for the treatment of Behçet´s disease with sight threatening posterior or panuveitis[END_REF] This gave us the idea to use IFN alpha also in patients with chronic treatment-resistant CME due to non-BD uveitis. Thus, in January 2003 we started to treat our first patient. In 2006 we reported for the first time promising results of this novel approach in eight patients. [START_REF] Deuter | Interferon alfa-2a: a new treatment option for long lasting refractory cystoid macular edema in uveitis? A pilot study[END_REF] After more than five years of experience, we now want to present a current update of our results on the efficacy and tolerability of IFN alpha treatment in patients with chronic treatment-resistant CME due to non-infectious uveitis.

Materials and methods

We present a retrospective analysis of an interventional case series that included 24 consecutive patients. To be eligible for IFN alpha treatment, patients had to suffer from chronic CME due to non-infectious uveitis in at least one eye. CME had to be resistant to pre-treatment consisting of systemic or intravitreal corticosteroids and/or immunosuppressives of adequate dose and duration. To qualify for analysis, patients had to complete follow-up of at least 3 months after initiation of IFN therapy. Informed consent was obtained from all patients before the start of treatment.

Depending on bodyweight, IFN alpha-2a (Roferon-A™, Hoffmann-La Roche) was administered at an initial dose of 3 million (mio) IU (≤ 70 kg) or 6 mio IU (> 70 kg) per day subcutaneously. In 10 patients IFN alpha-2a was then tapered stepwise over 6 months to a dose of 3 million IU twice a week and finally discontinued (on the basis of the dosing schedule we use in BD patients). In the case of a relapse of CME in at least one eye of the patient, IFN alpha-2a was reinstituted at a dose of 3 mio IU every second day with subsequent tapering in order to evaluate the lowest maintenance dose to maintain the absence of CME. In the remaining 14 patients a different treatment regimen was used that did not discontinue IFN alpha-2a after 6 months. Dosing schedules for IFN alpha-2a are shown in Figure 1. In all patients, when IFN treatment was inititated the dose of previous immunosuppressives was halved, acetazolamide was tapered and oral corticosteroids were reduced to a maximum of 10 mg prednisolone per day as quickly as possible. To initiate IFN treatment, patients were admitted to hospital for 3-4 days; further treatment was performed in an outpatient setting. To lessen flu-like symptoms, paracetamol was additionally administered in the initial treatment phase.

The main outcome criterion of the analysis was the efficacy of IFN treatment assessed as the response of CME which was measured by optical coherence tomography (OCT) at each visit. It was defined as:

• 'effective' if a complete resolution of CME was seen within 3 months and the dose of IFN alpha-2a could be tapered;

• 'partly effective' if an incomplete resolution of CME within 3 months was observed, the dose of IFN alpha-2a could not be tapered and/or acetazolamide could not be discontinued;

• 'not effective' if no response of CME was seen or a recurrence of CME occurred within 3 months.

As a secondary outcome criterion, development of visual acuity (VA) was measured in European decimals and converted to logMAR for computing. According to recommendations of the Standardization of Uveitis Nomenclature (SUN) Working Group, improvement of VA was defined as a decrease, and worsening of VA as an increase of at least 0.3 logMAR (= 3 lines). [START_REF] Jabs | Standardization of Uveitis Nomenclature (SUN) Working Group. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop[END_REF] To exclude contraindications but also to control side effects during IFN treatment, patients were seen by our group's internists before starting treatment and, if indicated, during follow-up. Routine blood parameters including complete blood count and liver enzymes were measured every 4-6 weeks.

Results

Characteristics and detailed results of patients are shown in Table 1. Twenty-four patients (9 male, 15 female) with a total of 40 affected eyes were analysed. The patients (25.0 % each). It has to be considered as not effective in only 5 eyes of 3 patients (12.5 % each). Pre-existing neutralizing auto-antibodies against IFN alpha (titre >1000 ng/ml) were most probably the reason for non-response in one of these patients (no. 8) and led to discontinuation of treatment after 1 month (the only patient in the series who, exceptionally, did not complete the required follow-up of 3 months).

Another of these 3 non-responding patients (no. 7) developed new auto-antibodies against IFN alpha with a recurrence of CME (IFN therapy stopped after 3 months) in both eyes.

Table 2 shows the development of mean foveal thickness and VA within the first 3 months of IFN treatment and, at the end of follow-up, relating to efficacy of IFN treatment. As shown in Figure 3, in the vast majority of patients CME responded very quickly to IFN treatment, demonstrating a remarkable decrease after 3 days and an almost complete resolution already within 2 weeks.

In 8 patients IFN therapy was discontinued after six months in the complete absence of CME. One patient (no. 6) was lost to follow-up then. In the remaining 7 patients CME relapsed, in 6 patients within 1-3 months and in 1 patient after 22 months.

Therefore IFN therapy had to be reinstituted, again demonstrating efficacy. Currently, 2 patients have been off IFN for 4 and 8 months, respectively, without recurrence of CME to date, after IFN treatment for 63 and 61 months, respectively. Six patients with a follow-up longer than 24 months (median 49 months, range 27-65 months) are in stable remission of CME at the timepoint of analysis with very low IFN doses between 0.5 mio IU every fifth day and 3 mio IU every third day.

In the effective and partly effective patients concomitant medication could be remarkably tapered during follow-up. Right at the start of IFN therapy, 14 patients were each on prednisolone, acetazolamide or immunosuppressives. Only 3 patients were undergoing no other therapy at initiation of IFN treatment. At the end of followup, concomitant medication included low-dose prednisolone in 12 patients. However, only 3 patients were still on acetazolamide, and only 5 patients on immunosuppressives. Seven patients did not need any other therapy. Discussion CME represents a sight-threatening complication of uveitis and therefore necessitates enhanced therapeutic efforts, especially if it becomes chronic. In our case series of patients with quiescent uveitis but long-lasting and refractory CME, IFN alpha was very effective, demonstrating remission or partial remission of CME in 87.5 % of patients or 87.5 % of eyes, respectively. We were surprised by the quick response of CME to IFN therapy. However, our data do not show any correlation between the grade of efficacy of IFN therapy and the duration of CME or the underlying uveitis condition (anterior, intermediate or posterior).

To date, it is unknown how long after the occurrence of CME an improvement in vision may still be possible, should an effective treatment be found. Hence, for us, the primary aim of IFN treatment was not to gain an increase in VA but to prevent patients from further loss of vision. In our analysis, out of 35 eyes, in which IFN treatment was demonstrated to be effective or partly effective, 34 eyes showed an improvement or at least maintenance of VA during follow-up compared to values before IFN therapy. As long as there are no exact parameters to predict visual development in eyes with chronic CME, in our opinion it is absolutely indicated to undertake therapeutic efforts as well in eyes with very long-lasting CME.

So far, we are unable to answer the question of whether and when IFN treatment can be discontinued without a relapse of CME. Our attempt to stop IFN therapy after six months was not successful. Perhaps this was too early. The question of IFN discontinuation was also hampered by the fact that several patients were not willing to stop IFN therapy because they were concerned about possible relapses of CME.

Probably, we have to consider that IFN alpha, similar to other immunomodulating drugs, will not be able to cure long-lasting CME. However, our results in patients with several years of follow-up demonstrate that it is possible to maintain the long-lasting absence of CME with very low IFN doses.

Although side effects occurred frequently, IFN treatment was generally well tolerated.

Most of the side effects were of mild to moderate severity and dose dependent. Only one patient decided to discontinue treatment. In the remaining patients, this was neither medically necessary nor desired by the patients. For them the advantages of the therapy clearly outweighed its side effects. Overall, we observed the same spectrum of side effects that has been reported in patients with BD treated with IFN alpha. [START_REF] Koetter | The use of interferon α in Behçet disease: Review of the literature[END_REF] However, the frequency of particular side effects, such as fatigue or increased liver enzymes, was higher in our patients than in those with BD. This may be due to the fact that, on average, our patients were significantly older. Whereas BD patients are usually between 20 and 40 years old at the start of IFN treatment, 50 % of the patients in our analysis were older than 50 years, 25 % even older than 66 years. Moreover, in several of our patients concomitant medication included mycophenolate mofetil which was not, as we do with imunosuppressives in BD patients, discontinued before initiation of IFN alpha and which may have contributed to increasing liver enzymes.

Only one patient (no. 8) had no side effects at all, including no flu-like symptoms, which represent the most common side effect of IFN alpha affecting more or less all patients after the first and second injection. In this patient we detected pre-existing auto-antibodies against IFN alpha. To date, we have no explanation for this phenomenon. However, it confirms our experience that the occurrence of flu-like symptoms after initiation of IFN therapy might be a positive sign with regard to the response of a patient. Thus, if a patient displays no flu-like symptoms at all or, as happened in patient no. 7, therapy ceases to have an effect after the initial response, anti-IFN auto-antibodies have to be excluded.

To date, the possible mode of action of IFN alpha in CME can only be hypothesized.

Keeping in mind the previous failure of anti-inflammatory and immunosuppressive drugs, the underlying mechanism of chronic uveitic CME is assumed to be not or not only inflammation but more probably a diffuse damage of the blood-retina barrier.

Thus the effect of IFN alpha we have seen here might be different from an only antiinflammatory or immunomodulating one. What we have clinically observed in our patients could correlate with the results of Gillies and Su, who in 1995 postulated that IFN alpha-2b, which is very similar to IFN alpha-2a, enhances the barrier function of bovine retinal microvascular endothelium in vitro. In their experiments IFN alpha-2b increased the electrical resistance in monolayers of bovine retinal capillary cells, decreased their permeability to inulin and enhanced their morphological differentiation. In contrast, IFN gamma did not demonstrate these effects. Thus, the authors hypothesized that IFN alpha might have therapeutic potential in diseases characterized by leakage of the vascular endothelium. [START_REF] Gillies | Interferon alpha-2b enhances barrier function of bovine retinal microvascular endothelium in vitro[END_REF] There are some factors which may limit the evidence of this study, such as its retrospective nature, the heterogeneity of the patients regarding the type of uveitis (anterior, intermediate or posterior) as well as the non-standardized dosing regimens for IFN and the concomitant medications.

In summary, our data demonstrate that IFN alpha is an effective and well-tolerated therapeutic option for long-lasting, otherwise refractory uveitic CME and therefore signifies a considerable advance in the treatment of such patients. However, there are still open questions that need to be answered in the near future regarding optimized dose regimens, co-medication, the possibility of discontinuation as well as better understanding of the exact mode of action. 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 on a worldwide basis to 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.

Competing Interest: None declared.

  median age of patients at the start of IFN treatment was 50.0 years (lower quartile (Q1) 42.0 years; upper quartile (Q3) 66.0 years; mean 51.0 years; range 19-74 years). Two-thirds of the patients suffered from bilateral CME. Localization of the uveitis was anterior in 2 patients (8.3 %), intermediate in 18 patients (75.0 %) and posterior (Birdshot chorioretinopathy) in 4 patients (16.7 %). An underlying systemic disorder was detectable in 1 patient with intermediate uveitis, who suffered from multiple sclerosis. Median duration of CME before IFN therapy was calculated for 36.0 months (Q1 24.8 months; Q3 85.0 months; mean 56.9 months; range 16-132 months). The uveitis itself was quiescent in all patients and did not necessitate any change or intensification of treatment at the timepoint when IFN was started. Ineffective pre-treatment for CME consisted of systemic corticosteroids in all patients, acetazolamide in addition in 22 patients and at least one additional immunosuppressive drug in 18 patients. Intravitreal drugs (triamcinolone and/or bevacizumab) were administered in 6 patients; in 9 patients surgical treatment of CME by pars plana vitrectomy had been performed. The median follow-up for all patients accounts for 21.0 months (Q1 11.8 months; Q3 42.8 months; mean 27.9 months; range 1-65 months).

Figure 2

 2 Figure 2 provides an overview of the efficacy of IFN treatment. This was shown as effective in 25 eyes of 15 patients (62.5 % each) and partly effective in 10 eyes of 6

  Side effects occurred frequently during IFN therapy. They included typical flu-like symptoms, especially after the first and second injection (23 patients), fatigue (10 patients), increased liver enzymes (9 patients), leucopenia (6 patients), alopecia (5 patients), transient detection of anti-thyroid auto-antibodies without developing clinical symptoms (2 patients), fibromyalgia and mild depression (1 patient each).Side effects were mostly dose dependent, improved during tapering of IFN alpha treatment, and led to its discontinuation in 1 patient only (no. 16; patient´s personal decision due to persisting fatigue).

Patient

Table 1 : 2 :

 12 Abbreviations: AU = anterior uveitis; IU = intermediate uveitis; PU = posterior uveitis; Ster = steroids; Acet = acetazolamide; MMF = mycophenolate mofetil; CsA = cyclosporine A; MTX = methotrexate; Aza = azathioprine; Cyclo = cyclophosphamide; Triam = triamcinolone; Bevac = bevacizumab; ppV = pars plana vitrectomy; Pred = prednisolone; OD = right eye; OS = left eye; OU = both eyes; E = effective; PE = partly effective; NE = not effective; Flu = flu-like symptoms; Fat = fatigue; Alop = alopecia; Liv = increased liver enzymes; Leuk = leucopenia; Fibro = fibromyalgia; Ant = auto-antibodies; Depr = depression; NAV = not available; NAP = not applicable.

Figure 1 :

 1 Figure 1: Dosing regimens for IFN alpha-2a.

Figure 2 :

 2 Figure 2: Overview of the efficacy of IFN treatment in the 24 patients.

Figure 3 :

 3 Figure 3: Development of foveal thickness in the 40 eyes.

Table 1 Mean foveal thickness and range (µm) Change in VA (no. of eyes) compared to initiation of IFN treatment

 1


Dr Johannes Schwartzkopff
email: johannes.schwartzkopff@uniklinik-freiburg.de

Moritz Berger

Florian Birnbaum

Daniel Böhringer

Thomas Reinhard

Accelerated Corneal Graft Rejection in Baby Rats

Keywords: Baby rat, graft rejection, penetrating keratoplasty, NK cell

come   L'archive ouverte pluridisciplinaire

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.

Introduction

Keratoplasty can restore corneal clarity in most cases. This is especially important in early childhood during which any form of uncorrected corneal opacity or refractive error leads to life-long poor vision (amblyopia). However, while adults show a very low rate of corneal graft rejection in standard situations of only 10%, corneal graft failure occurs in up to 85% of transplanted children under 3 years of age [START_REF] Althaus | Keratoplasty in newborns with Peters' anomaly[END_REF][START_REF] Reidy | Penetrating keratoplasty in infancy and early childhood[END_REF][START_REF] Yang | Long-term results of corneal graft survival in infants and children with peters anomaly[END_REF]. Astonishingly, this is observed with no sign of preexisting ocular surface inflammation, as only seen otherwise in high risk corneal beds.

Here infection, chemical burn, immunological disorders such as ocular pemphigoid, perforation or re-transplantation lead to an elevated rejection rate. Thus, circumstances other than a preinflamed eye seem to be responsible for graft failure in infant humans. It is of great interest to gain insight into the underlying mechanisms to prevent these children from becoming amblyopic.

Various animal models revealed evidence that CD4 + T cells play a predominant role during graft rejection in low and high risk corneal beds [START_REF] Niederkorn | Differential roles of CD8+ and CD8-T lymphocytes in corneal allograft rejection in 'high-risk' hosts[END_REF][START_REF] Yamada | Role of CD4+T cells in immunobiology of orthotopic corneal transplants in mice[END_REF]. These animal models vary in their dynamics and strength of immune reactions but reproduce the clinical results with respect to corneal graft survival. In short, Streilein and others demonstrated that the excellent survival rates following keratoplasty are a consequence of the immune privileged status of the cornea and the eye itself [START_REF] Streilein | Ocular immune privilege: therapeutic opportunities from an experiment of nature[END_REF][START_REF] Niederkorn | Immune mechanisms of corneal allograft rejection[END_REF].

Our group proposed an animal model for keratoplasty in baby rats to address this problem in the young. However, nothing had yet been reported on the rejection kinetics or mechanisms underlying graft failure in these young animals [START_REF] Mayer | Differential contribution of natural killer cells to corneal graft rejection in 3-week-old versus mature rats[END_REF]. We used this model to elucidate the factors that might lead to immunological graft rejection in young rats.

Materials and Methods

Animals and Anesthesia

Inbred female Fisher (Rt1 lv1 ) and Lewis (Rt1 l ) rats (Charles River, Germany) were used as donors and recipients. All animals were treated in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rats were anesthetized with a short inhalation of isofluran followed by an intraperitoneal injection of a mixture of ketamine, xylazine and atropine, respectively.

Corneal transplantation

Orthotopic penetrating keratoplasties were performed as described previously [START_REF] Mayer | Differential contribution of natural killer cells to corneal graft rejection in 3-week-old versus mature rats[END_REF][START_REF] Birnbaum | The new malononitrilamide immunosuppressant FK778 prolongs corneal allograft survival in the rat keratoplasty model[END_REF]. Briefly, Fisher donor buttons (2.5mm) were obtained and the animals were killed thereafter. Recipients were anesthetized and the central cornea was removed using a 2mm trephine. The graft was fixed with 8 interrupted sutures (11.0 Ethilon, Ethicon, Germany). Finally, a blepharoraphy was applied. To exclude any potential difference in the graft's immunogenicity due to its size and the vicinity to peripheral antigen presenting cells (APC) in young recipients, the protocol was modified for 10 week old recipients and a corneal button with a diameter of 3.5mm was orthotopically transplanted.

Clinical graft assessment and rejection kinetics

All grafts were daily examined by two investigators for signs of opacity according to an international score [START_REF] Birnbaum | The new malononitrilamide immunosuppressant FK778 prolongs corneal allograft survival in the rat keratoplasty model[END_REF][START_REF] Claerhout | Role of natural killer cells in the rejection process of corneal allografts in rats[END_REF]. Grafts with technical difficulties such as cataract, infection, loss of the anterior chamber or massive hyphema were excluded. The time point of rejection was identified as an opacity score of 4 (complete opacity).

Groups

Only 10-week-old donor buttons were used considering the fact that in humans most of the grafts are derived from adults. Group 1 consisted of 10-week-old Fisher donor and Lewis recipients. In group 2, a 10-week-old Fisher cornea was used and grafted to a 3-week-old Lewis recipient. Groups 3 and 4 resembled the syngeneic controls. Each group constisted of 12 recipients.

Immunohistochemistry

Cryosections of four bulbi per group were taken and fixed in acetone. Primary mouse-anti-rat antibodies were applied and a biotinylated rabbit anti-mouse secondary antibody was used to bind a streptavidine alcaline phosphatase. After incubation with a substrate (alkalinephosphatase-kit1, Vector, USA), sections were counterstained with Mayer's hematoxylin.

Positively stained cells were counted within three squares in the grafted corneal stroma and calculated as mean cellular infiltrate per mm 2 .

Antibodies

Primary antibodies used for immunohistochemistry were anti-CD4 (clone W3/25), anti-CD8 (clone OX-8), anti-CD161 (clone 10/78), anti-CD163 (clone ED2), anti-dendritic cells (clone OX-62) and anti-CD25 (clone Ox-39). A secondary biotinylated polyclonal rabbit-anti-mouse immunoglobulin and streptavidine alkaline phosphatase were obtained from DAKO, Germany.

Statistics

The time interval from corneal transplantation to rejection was analyzed using the Kaplan-Meier method and the groups compared using the log-rank test. The densities of infiltrating immune cells were compared statistically using the T-test.

Results

1) Complete rejection occurs earlier in young than in old recipients

All of group 1's grafts failed after a median rejection time of 15 days. Group 2's transplants were rejected after a median period of only 9 days (fig. 1a). The difference in the survival rates between groups 1 and 2 proved to be statistically highly significant (p<0.01). None of the syngeneic controls rejected. We transplanted in 10 week old Lewis rats a 3.5mm corneal button to a 3.0mm corneal bed to rule out that the observed difference in graft survival was a consequence of the diverse ratio between donor and recipient corneal tissue and of the different distance of the graft's margin to the limbal APC between young and old animals, respectively. As apparent in fig. 1b, we saw no statistical difference, regardless of whether a 2.5mm (group 1) or a 3.5mm corneal button (group 1a) had been grafted.

2) The time point of complete rejection coincides with a lower cellular infiltrate in young recipients On the median days of graft failure (days 9 and 15) we analyzed infiltrating mononuclear cells on HE-stainings. A moderate cellular infiltrate was detected in both syngeneic control groups (fig. 2). Yet we also observed a remarkable infiltration of leukocytes in both allogeneic groups 1 and 2.

However, the density of cells within the graft proved to be statistically significantly higher in the group 1 animals on the day of rejection (day 15) when compared to group 2 at the equivalent time point (day 9) (p<0.0001).

3) Accumulation of cells of the adaptive immunity in the graft

Infiltrating cells were stained in rejected corneas. Calculations of infiltrating CD4 + and CD8 + T cells, activated CD25 + cells and Ox-62 + dendritic cells are shown in figs.3a-d. No significant cellular infiltration was seen in the syngeneic controls. Hardly any cells were stained positively on day 9 in group 1's grafts, whereas we detected a clear infiltrate in the transplants of group 2. This difference was statistically significant (p<0.01). However, by day 15 all cell types had increased, that no significant differences in cell numbers were detectable between groups 1 and 2 any more. No further increase in cellular infiltration was observed in both groups thereafter. On the respective median days of rejection (i.e. day 9 for young and day 15 for old recipients) CD4 + , CD8 + and Ox-62 + cells infiltrated statistically significantly stronger in group 1's animals (p<0.01), whereas no siginficant difference was observed for CD25 + cells.

4) Infiltration of cells of the innate immune systeme

Infiltrating CD163 + macrophages and CD161 + NK/NKT cells were stained on the median time points of rejection (days 9 and 15). Both cell types were low in cell numbers on day 9 in group 2, whereas nearly no infiltration was visible in group 1 (p<0.01). CD163 + macrophages accumulated in both groups that no statistical difference was visible on day 15 any more (fig. 4b).

On the respective days of rejection (i.e. day 9 for group 2 and day 15 for group 1) statistically significantly more CD163 + macrophages infiltrated in old recipients when compared to young animals (p<0.01). CD161 + NK/NKT cells increased on day 15 in groups 1 and 2, but dominated statistically significantly in group 2's animals when compared to the group 1 (p<0.01) (fig. 4c).

No statistical difference could be seen for infiltrating CD161 + cells when comparing the median time points of rejection of the respective group (i.e. day 9 for group 2 and day 15 for group 1).

Discussion

It is known from humans that graft failure in children under 3 years of age occurs more frequently than in adults [START_REF] Althaus | Keratoplasty in newborns with Peters' anomaly[END_REF][START_REF] Reidy | Penetrating keratoplasty in infancy and early childhood[END_REF][START_REF] Yang | Long-term results of corneal graft survival in infants and children with peters anomaly[END_REF]. It is thus imperative to improve graft survival, because any form of opacification or refractive error of the cornea leads to amblyopia. In order to be able to specifically treat children following penetrating keratoplasty, it is important to better understand the mechanisms that lead to immunological graft failure. Our group recently suggested a rat model for corneal transplantation that enabled us to study the rejection of corneal allografts in young individuals [START_REF] Mayer | Differential contribution of natural killer cells to corneal graft rejection in 3-week-old versus mature rats[END_REF]. However, nothing so far was reported on the dynamics of graft rejection in young individuals.

Kinetics of corneal allograft failure

Fig. 1 illustrates that rejection kinetics appeared to differ between 3-and 10-week-old recipients.

Rejection did appear significantly earlier in 3-week-old than in 10-week-old recipients (day 9 vs. day 15). Corneal antigen presenting cells (APCs) play an important role during immunological corneal allograft failure and are located mostly in the periphery nearby the limbus [START_REF] Liu | Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts[END_REF][START_REF] He | Depletion of donor-derived Langerhans cells promotes corneal allograft survival[END_REF][START_REF] Hamrah | Corneal immunity is mediated by a heterogenous population of antigen-presenting cells[END_REF]. The diameter of the 10-week-old rat cornea is significantly larger than that of a 3-week-old animal.

Therefore, the margin of a corneal graft gets closer to these corneal APC in a young recipient and the increased rejection could be seen as a consequence of an earlier onset of an allo-recognition by these APCs. To exclude this, the graft's diameter was elevated in old recipients. However, the median graft survival time was not changed in our model (fig. 1b) and thus the vicinity of the graft to the peripheral APCs did not influence the rejection process. Our findings are in contrast to common clinical observations where a larger graft correlates with a higher incidence for rejection [START_REF] Katami | The mechanisms of corneal graft failure in the rat[END_REF]. In our model a single minor mismatch induces a devastating allograft response within the first 15 postoperative days. In this short period graft size does not seem to be a risk factor and might be overrun by the aggressive immune response in this model.

A stronger lymphangiogenesis has been reported in 8-week-old when compared to 15-months-old mice and was suggested to influence a stronger corneal allograft failure in young animals [START_REF] Bock | Improved semiautomatic method for morphometry of angiogenesis and lymphangiogenesis in corneal flatmounts[END_REF].

However, this rather reflects a reduced corneal lymphangiogenesis in aged animals. It would be necessary to analyze the density of lymphatics in 3-week-old compared to 8-week-old after an inflammatory stimulus to understand their role during the elevated allograft rejection in our model. However, lymphangiogenesis influences corneal allograft rejection and it certainly might be an important factor for corneal allograft rejection in the baby rat model.

Corneal graft failure coincides with low immune infiltration

Astonishingly, the rejected grafts of young recipients presented fewer infiltrating cells than did these of the older recipients at the time of immunological graft failure (fig. 2). Considering these data together with our observation that graft failure in young animals occurred more vigorously, we wondered what rendered those few infiltrating immune cells within the transplant so potent that they could lead to graft failure. Several cell types have been reported to contribute to corneal allograft rejection [START_REF] Niederkorn | Immune mechanisms of corneal allograft rejection[END_REF]. However, the major player turned out to be CD4 + T cells [START_REF] Yamada | Role of CD4+T cells in immunobiology of orthotopic corneal transplants in mice[END_REF]. Together with the knowledge that the T cell system develops within early childhood [START_REF] Kovarik | Immunity in early life[END_REF][START_REF] Adkins | Neonatal adaptive immunity comes of age[END_REF], one possible explanation is that a specific T cell response in young recipients is missing. We compared the T cell pool of 3-and 10-week-old Lewis rats and observed no differences in the numbers of various T cell populations, nor in the CD4/8 T cell ratio in blood, spleen and lymphnode (not shown).

Thus, a fully-developed T cell system is probable.

Characterization of the infiltrate within a corneal allograft

We were able to characterize the immune response leading to allograft failure in greater detail by staining for a variety of leukocyte populations such as CD4 + and CD8 + T cells, CD25 + cells, CD163 + macrophages and Ox-62 + dendritic cells on the median time points of rejection (days 9 and 15). In 10-week-old rats hardly any of these cells infiltrated whereas a significant cellular infiltrate was observed in 3-week-old old recipients on day 9. (figs. 3+4). An accumulation of all of these cells was observed until day15 in both groups without an additional increase thereafter. This demonstrates that an unspecific additional inflammation was present in 3-week-old recipients even after they had experienced complete graft failure. However, other factors more unspecific than T cells might have contributed to complete opacity in young recipients. It is known, that NK cells play a pivotal role in compensating for an underdeveloped T cell system in young individuals [START_REF] Kirwan | Regulation of natural killer cell activity[END_REF][START_REF] Freud | Human natural killer cell development[END_REF]. However, aside from similar T cell numbers in 3-and 10-week-old Lewis rats, we noted no differences in the numbers of CD3 -CD161 + NK and CD3 + CD161 + NKT cells between the two recipient groups in blood, spleen and lymphnode (not shown).

Differences in infiltrating NK/NKT cells

Histological staining on postoperative day 9 revealed a statistically significant dominance of infiltrating CD161 + NK/NKT cells in young versus old recipients. Although we were able to also demonstrate an accumulation of CD161 + cells on day 15 in both groups, this did not equalize the cell numbers. In fact, CD161 + NK/NKT cells were still statistically significantly higher in cell numbers on postoperative day 15 in young recipient rats (fig. 4c). However, unlike T cells, CD161 + cells infiltrated to a similar extend on the actual days of rejection for the respective groups (i.e. day 9 for the 3-week-old recipients and day 15 for the 10-week-old recipients). We therefore hypothesize that NK/NKT cells in young recipients have a stronger potency to reject a corneal graft, which might be due to the cells theirselves or to age-dependent changes in the suppressive ocular environment between young and old rats. This led us to the conclusion that CD161 + cells might be a key factor during corneal allograft failure in 3-week-old recipients.

It has been reported that NK cells play a role during corneal graft rejection in rats [START_REF] Claerhout | Role of natural killer cells in the rejection process of corneal allografts in rats[END_REF] and that NKT cells contribute to the induction of anterior chamber associated immune deviation (ACAID) [START_REF] Sonoda | Long-term survival of corneal allografts is dependent on intact CD1d-reactive NKT cells[END_REF]. Both NK as NKT cells contribute to the innate immune system and may be key to immune mediated pathologies in young individuals. Depletion experiments [START_REF] Van Den Brink | In vivo treatment with monoclonal antibody 3.2.3 selectively eliminates natural killer cells in rats[END_REF] of either of the two CD161 + cell populations, or transplantation into nude rats lacking T and B cells but owning a competent NK cell system [START_REF] Rolstad | The athymic nude rat: An animal experimental model to reveal novel aspects of innate immune responses?[END_REF] would give better insight into the role of infiltration NK and NKT cells, respectively. Furthermore, additional analyses of the aqueous humor [23] or cytokines within a rejected graft [24] would lead to better understanding of the processes and of the cells that contribute to graft failure in infants.

Considering these data together, we have demonstrated for the first time an animal model of keratoplasty that mimics the faster rejection of corneal allografts in human infants. In so doing, we made investigation of an allogeneic immune reaction after keratoplasty in infants possible.

Although an underdeveloped T cell system in our model seems to be unlikely, we demonstrated that CD161 + NK/NKT cells may be key players in corneal allograft rejection in young individuals. Even if additional congenital or structural abnormalities count as poor prognistic factors in humans infants regarding corneal allograft rejection [25], our observation may ultimately lead to a better understanding of the mechanisms of graft failure in human infants and provide new treatment strategies for its prevention. Median survival times were compared in 10 week old recipients using grafts of two different diameters (i.e. 2.5mm onto a 2.0mm corneal bed and 3.5mm onto a 3.0mm corneal bed, respectively). No significant difference in median survival times could be observed between rejection of a corneal transplant with a diameter of 2.5mm (1) and 3.5mm (1a) (p>0.1). The infiltration of CD163 + cells was statistically significantly elevated in animals of group 1 on the median rejection time point (day15) when compared to group 2 on its median rejection time point (day9) (p<0.01), whereas no statistical difference was observed for CD161 + cells. The infiltration of CD4 + , CD8 + and Ox-62 + cells was statistically significantly elevated in animals of group 1 on the median rejection time point (day15) when compared to group 2 on its median rejection time point (day9) (p<0.01), whereas no statistical difference was observed for CD25 + cells.

Funding: 1 2 Fig. 1 :

 121 Fig. 1: a) Kaplan-Meier analyses of graft survival. Animals of group 1 (10 week

Fig. 2 :

 2 Fig.2: Mean cellular infiltration per mm 2 within the graft on the expected days of rejection

Fig. 3 :

 3 Fig. 3: Mean cellular infiltration was calculated from 4 individual corneal transplants on the

Fig. 4 :

 4 Fig. 4: Mean cellular infiltration of CD163 + macrophages and CD161 + NK/NKT cells was

Fig. 1 :Fig. 2 :

 12 Fig. 1: a) Kaplan-Meier analyses of graft survival. Animals of group 1

Fig. 3 :

 3 Fig. 3: Mean cellular infiltration was calculated from 4 individual corneal transplants

Fig. 4 :

 4 Fig. 4: Mean cellular infiltration of CD163 + macrophages and CD161 + NK/NKT cells

Acknowledgments:

We thank Dr. Hanspeter Pircher and Dr. Laura Bredow for helpful discussion, Carole Cürten for helping with the manuscript, Linda Sipp for excellent animal husbandry and the Ernst-und-Berta Grimmke foundation, Duesseldorf, Germany for supporting this project.

Footnotes

This work was supported by the Ernst-und-Berta Grimmke Stiftung, Düsseldorf, Germany.

None of the authors has a conflict of interest.

Abbreviations