Zia I Carrim
email: zia.carrim@doctors.org.uk

John Richardson

William N Wykes

Incidence and visual outcome of acute endophthalmitis after cataract surgery -Experience of a single eye department in Scotland

Aim

Endophthalmitis remains one of the most dreaded complications of modern cataract surgery. Its nationwide incidence has been estimated but published data on accurate incidence at regional level is scarce. This audit examines incidence and visual outcome of endophthalmitis from a single eye department in Scotland over a 7 year period. Findings are compared with those from other series.

Methods

A retrospective consecutive audit of all cases of acute endophthalmitis treated between 1 st January 2000 and 31 st December 2006 at the Southern General Hospital in Glasgow was undertaken. Details of each case in relation to presentation, treatment and outcome of endophthalmitis were recorded. Cross-tabulations were performed to identify prognostic factors of visual outcome.

Results

Twenty-five patients were treated for endophthalmitis over the audit period. The incidence of endophthalmitis was 0.20% (95% confidence interval, 0.10-0.30%). There were 12 (48%) culture positive cases with coagulase negative staphylococcus accounting for 58%. After treatment, sixteen patients (64%) achieved driving standard vision or better. Poor vision at presentation and streptococcal endophthalmitis were associated with poor visual outcome.

Conclusion

The incidence of endophthalmitis in this series is comparable to larger studies suggesting that accepted benchmarks, despite being estimates, reflect UK practice. Visual outcome, with treatment, can be favourable in a significant proportion of patients.

Introduction

Advances in cataract extraction over the last decade, driven in part by the transition from extracapsular to phacoemulsification surgery, have made this intervention one of the most frequently performed in the United Kingdom (UK) [START_REF]Headline figures[END_REF] . Approximately 300 000 operations are carried out annually amidst high public expectations of good visual outcome and speedy recovery [START_REF]Headline figures[END_REF] . Post-operative endophthalmitis remains one of the most visually devastating complications of cataract surgery. In the UK, national standards have been established via the Department of Health funded National Cataract Surgery Surveys (NCSS) in 1990 and 1998 [START_REF] Desai | The national cataract surgery survey: Clinical outcomes[END_REF][START_REF] Desai | National cataract surgery survey 1997-8. A report of the results of the clinical outcomes[END_REF] .

The British Ophthalmological Surveillance Unit (BOSU) also runs a prospective surveillance programme for all cases of post-operative endophthalmitis via its monthly reporting card system.

An incidence rate of 0.14%, with accompanying epidemiological data, was reported in 2004 based on a surveillance period from October 1999 to September 2000 [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . One of the observations of the BOSU survey was that few centres in the UK kept records of postoperative infections. It was also estimated that only 62% of cases of post-operative endophthalmitis were reported [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] .

The eye department at the Southern General Hospital (SGH) in Glasgow has an electronic database which allows patients to be retrospectively identified based on diagnosis. This database was used to carry out a retrospective audit of the incidence and visual outcome of acute endophthalmitis after cataract surgery. It was felt that data from a single UK eye unit would complement the interpretation of the BOSU incidence rate which is widely regarded as a nationwide benchmark.

Methods

All patients treated for acute post-operative endophthalmitis at the SGH between 1 st January 2000 and 31 st December 2006 were identified from an electronic register. For the purposes of this audit, a clinical diagnosis of presumed endophthalmitis was defined as excessive postoperative intraocular inflammation irrespective of subsequent microbial culture results or steroid responsiveness. Only patients who developed endophthalmitis after undergoing cataract surgery as a single procedure were included. Casenotes were reviewed with specific attention to details of surgery, presentation of endophthalmitis, treatment and final visual outcome. When present, systemic comorbidities relevant to the risk of endophthalmitis were also recorded (diabetes, malignancy, immunosuppression, recurrant chest or urinary tract infections). The final visual outcome was defined as the best vision achieved after treatment and maintained over at least 2 consecutive visits 4 weeks apart.

A grading system for final visual outcome was devised based on three categories -good (final best corrected visual acuity (BCVA) better than pre-op BCVA and better than 6/60), fair (final BCVA worse than the pre-op BCVA but better than 6/60), and poor (final BCVA worse or equal to 6/60). The threshold of useful vision was chosen to be 6/60. To study the impact of poverty on visual outcome, post-code based Carstairs deprivation scores (CDS) were derived for each patient and converted to 3 categories -not deprived (CDS < 0), deprived (0 ≤ CDS < 4) and very deprived (CDS ≥ 4) [START_REF] Mcloone | Carstairs scores for Scottish postcode sectors from the 2001 census[END_REF] .

Data on the number of cataract operations performed each year were obtained from the Department of Audit and Clinical Effectiveness and used to calculate annual incidence figures for post-operative endophthalmitis. All patients having had cataract surgery at the SGH would, upon developing endophthalmitis, have presented or been referred back to us for management.

An accurate determination of incidence was therefore possible within the framework of the National Health Service (NHS). Statistical measures of concordance for ordinal categories (Pearson's r, Spearman's rho, Somers' d, Kendall's tau-b, Goodman-Kruskall gamma) were applied to our grading system of final visual outcome and used to identify potential prognostic factors. All calculations were performed using MINITAB 14.

Results

Over the 7 year period of this retrospective consecutive series, 25 cases of post-operative endophthalmitis, meeting the inclusion criteria, were treated at the SGH. Nineteen (76%) had their cataract surgery at the SGH with the remaining 6 (24%) having theirs elsewhere. There were 15 (60%) males and 10 (40%) females with an overall mean age of 72 ± 12 years. The mean CDS was 1.2 ± 5.5. Sixteen patients (64%) had at least one relevant systemic comorbidity with 6 (24%) being diabetic. All had pre-operative skin preparation using povidone iodine. A local anaesthetic technique was employed in all but one. Twenty three (92%) patients had phacoemulsification via clear corneal incision and 2 (8%) were converted to extracapsular cataract extraction (ECCE) after attempted phacoemulsification. Surgery was uncomplicated in 21 (84%) with the remaining 4 (16%) having posterior capsular (PC) rupture and vitreous loss.

All patients received post-operative chloramphenicol and prednisolone acetate drops. The mean time from surgery to presentation with endophthalmitis (PwE) was 4.2 ± 2.9 days (median 3, range 1-14). The visual acuity at presentation was 6/60 or worse in 20 (80%) patients. Seven (28%) patients were presumed to have a low grade endophthalmitis at presentation. The presence or absence of a relative afferent papillary defect (RAPD) was documented in only 11 (44%) cases and was clearly positive in 5 (45%).

A vitreous biopsy with injection of intravitreal antibiotics was undertaken in 20 (80%) patients. A sample of aqueous from the anterior chamber (AC) was also taken in 16. Two (8%) patients, with presumed low-grade endophthalmitis, had AC samples taken only. Three (12%) patients had neither a vitreous biopsy nor an AC tap -two on account of low-grade endophthalmitis and one for medical reasons (multiple co-morbidities and general frailty). Vitreous biopsy yielded an organism in 12 (60%) patients. Growth on an appropriate culture medium was heavy in 5 (42%), moderate in 2 (17%) and light in 5 (42%). There were 7 (58%) isolates of coagulase negative staphylococcus, 4 (33%) streptococcus species in general and 1 isolate identified only as gram positive cocci. Of the 18 AC samples, only 4 (22%) yielded an organism. The vitreous biopsy in these 4 cases was invariably positive for the same organism.

Information on the specific regime of intravitreal antibiotics was verifiable for 17 of the 20 patients. Vancomycin and amikacin were given in 7 (41%) patients whereas vancomycingentamicin, vancomycin-cefuroxime and cefuroxime-gentamicin combinations were given in 5 (29%), 2 (12%) and 3 (18%) patients respectively. All 25 patients in this series had intensive topical antibiotics. Eleven (44%) patients had oral steroids. A vitrectomy was undertaken in 5 (20%) patients -3 had perception of light (PL) vision at presentation and 2 had hand movements (HM) vision.

The mean time to achieving a final visual outcome was 3.8 ± 3.4 months (range 0.5 -13).

Based on our classification, the final visual outcome was good in 16 (64%) patients, fair in 4 (16%) and poor in 5 (20%). Of those with a poor outcome, 2 patients had a comfortable eye with no perception of light and 1 required an enucleation for a blind painful eye. There was a statistically significant association between poor vision at presentation and a poor visual outcome (p=0.018). In culture-positive vitreous biopsies, coagulase negative staphylococcus was associated with a better final visual outcome than streptococcus species (p=0.01). In this series, there was no statistically significant relationship between deprivation and visual outcome (p= 0.95). Presence of systemic comorbidity (p=0.76), complicated surgery (p= 0.97), presence of RAPD (p=0.12), timing of PwE (p=0.90), positive vitreous biopsy (p=0.21), heavy growth in culture-positive vitreous biopsy (p=0.89), intravitreal antibiotic regime (p=0.20) and oral steroid use (p=0.60) were similarly not found to be statistically significant predictors of final visual outcome (table 2). Between 1 st January 2000 and 31 st December 2006, 9664 cataract extractions were undertaken at the SGH. The overall incidence of endophthalmitis was 0.20% (95% confidence interval, 0.10% -0.30%). Only cases of endophthalmitis following surgery at the SGH were included in the calculation.

Discussion

Our incidence of 0.20 % for post-operative endophthalmitis is comparable to the corrected estimate from the BOSU survey and arguably higher than other UK studies (table 3) 3, 4, 6 -8 . This can be explained by the broad case definition applied in order to address previous concerns about under-reporting [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . Since endophthalmitis can lead to rapid loss of vision, excessive inflammation, particularly in the early post-operative phase, should be regarded as infective endophthalmitis. In our series, 28% (7/25) patients were deemed to have an acute low grade endophthalmitis at initial presentation reflecting the difficulty in distinguishing between a predominantly inflammatory rather than infective aetiology. Microbiological investigations in these patients, when undertaken, were also negative but it is known that conventional culture techniques do not indentify all infective cases [START_REF] Therese | Polymerase chain reaction in the diagnosis of bacterial endophthalmitis[END_REF] . Cases of presumed acute low grade endophthalmitis, which could in retrospect be labelled as inflammatory, have, therefore, been included to provide an accurate reflection of our clinical practice.

There appears to be little regional variation in the microbiological profile of endophthalmitis in the UK. In this series, samples for microbiology were obtained in 88% (22/25) of patients and were positive in 55% (12/22). Vitreous biopsies were positive in 60% (12/20) whereas AC taps were positive in 22% (4/18) only. There was no instance of a positive AC tap with a negative vitreous biopsy in the same patient. Gram positive organisms accounted for all positive cultures with coagulase negative staphylococci being identified in 58% (7/12). These findings are consistent with the BOSU survey and a recent study by Mollan et al in the West Midlands (table 4) [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF][START_REF] Mollan | Postcataract endophthalmitis: incidence and microbial isolates in a United Kingdom region from 1996 through 2004[END_REF] . Whilst the importance of an AC tap may not be apparent in this series, about 7% of patients who had both an AC tap and a vitreous biopsy in the BOSU survey and the study by Mollan et al had a positive culture from aqueous only [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF][START_REF] Mollan | Postcataract endophthalmitis: incidence and microbial isolates in a United Kingdom region from 1996 through 2004[END_REF] . It was also observed in the BOSU survey that pseudomonas was cultured from aqueous but not vitreous in 60% of reported pseudomonas isolates [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . An AC tap can therefore be valuable in indentifying true infective cases of endophthalmitis and should be performed in all presumed cases [START_REF]ESCRS guidelines on prevention, investigation and management of post-operative endophthalmitis[END_REF] . In our series, only 72% (18/25) had AC taps. This is a possible explanation for the absence of gram negative isolates.

The vancomycin/amikacin combination of intravitreal antibiotics was the most popular choice in our series (41%) as well as the BOSU survey (55%). The use of this combination in the Endophthalmitis Vitrectomy Study (EVS) may explain its popularity [START_REF]Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study[END_REF] . Overall, 80% (20/25) of our patients received intravitreal antibiotics in comparison with 96% in the BOSU survey. Of those that did not in this series, 80% (4/5) were presumed to have a low grade endophthalmitis with 3 (60%) having a visual acuity of 6/24 or better at presentation. These patients were treated with intensive topical antibiotics and steroid and, in each case, had a final visual outcome which was deemed good. In the BOSU survey, 75% of patients who did not receive intravitreal antibiotics had 6/60 vision or worse. It is arguable that presumed acute low grade endophthalmitis poses a management dilemma being indistinguishable from a severe sterile uveitis and not having all the features of a full-blown aggressive infective endophthalmitis. It is also possible that some cases may represent manifestations of toxic anterior segment syndrome (TASS) [START_REF] Holland | Update on toxic anterior segment syndrome[END_REF] . To our knowledge, there are no guidelines applicable to the management of presumed acute low grade endophthalmitis. Neither the EVS nor the BOSU survey specifically alludes to this small but significant proportion of patients in whom the risks of intravitreal therapy may even outweigh the intended benefits [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF][START_REF]Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study[END_REF] .

In the pre-EVS era, the importance of pars plana vitrectomy (PPV) in the management of endophthalmitis was unclear [START_REF] Doft | Treatment of postcataract extraction endophthalmitis[END_REF] . It was subsequently demonstrated that patients who present with perception of light (PL) vision benefit most from immediate vitrectomy [START_REF]Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study[END_REF][START_REF] Doft | Treatment of postcataract extraction endophthalmitis[END_REF] . Strict inclusion criteria were used in the EVS such that the findings, it can be argued, apply to very specific subgroups of patients [START_REF] Doft | Treatment of postcataract extraction endophthalmitis[END_REF][START_REF] Maguire | Postoperative endophthalmitis: Optimal management and role and timing of vitrectomy surgery[END_REF] . Over the years, indications for PPV in the management of postoperative endophthalmitis have evolved and also been subject to varied interpretations [START_REF] Maguire | Postoperative endophthalmitis: Optimal management and role and timing of vitrectomy surgery[END_REF] . It is frequently reported that, thanks to the EVS, more patients are now managed in the office setting with vitreous tap and intravitreal antibiotics [START_REF] Flynn | Legacy of the Endophthalmitis Vitrectomy Study[END_REF] . It can be inferred that the threshold for PPV varies significantly on either side of the Atlantic. The BOSU survey finding of 70% of patients with PL vision not undergoing PPV suggests that UK ophthalmologists have adapted the EVS recommendations to their local context [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . It is possible that the difficulty in accessing a vitreoretinal service, often provided from a tertiary referral centre, may account for a much higher threshold for immediate PPV. The inclination of most UK ophthalmologists may be to try intravitreal antibiotics first. In this series, 20% (5/25) of all patients and 75% (3/4) of those who presented with PL vision had a PPV despite there not being a vitreoretinal service on-site. Since our series is more recent, it may also reflect changing attitudes amongst UK ophthalmologists.

In this series, 80% (20/25) of patients had a presenting visual acuity of 6/60 or worse and 64% (16/25) achieved driving standard vision or better after treatment. In the BOSU survey, 85% had a visual acuity of 6/60 or worse and 48% achieved driving standard vision or better [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . We felt the need to devise a scale to grade visual outcome in order to give due consideration to preoperative vision as this was likely to influence patient perception of visual recovery. According to this scale, the final visual outcome was better than pre-operative vision in 64% (16/25). When using an acuity of 6/60 as a threshold for functional vision, 80% (20/25) achieved this or better in this series in comparison with 66% in the BOSU survey [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . It is difficult to attribute better visual outcome to any one variable. However, it is possible that a greater awareness of the symptoms of endophthalmitis amongst our patients is reflected in earlier presentation (95.6% within 9 days versus 95% within 18 days in the BOSU survey [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] ). Earlier treatment may account for better outcome.

The EVS has shown that older age, diabetes, corneal infiltrates, PL vision, rubeosis and a ruptured PC are all independent risk factors for decreased final visual acuity [START_REF]Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study[END_REF] . It has also been shown that visual prognosis is strongly associated with the type of infecting organism [START_REF]Endophthalmitis Vitrectomy Study Group. Mircobiologic factors and visual outcome in the Endophthalmitis Vitrectomy Study[END_REF] .

Although a rigid analysis of prognostic factors for visual outcome was beyond the scope of this small series, some known trends have been replicated. Visual acuity at presentation was a statistically significant predictor of visual outcome with all patients presenting with PL vision having a poor outcome. In culture positive cases, infection with streptococcus sp was also significantly associated with poor outcome. It is argued that the recommendations of the EVS do not apply to streptococcal endophthalmitis for which an immediate vitrectomy, irrespective of vision, is beneficial [START_REF]ESCRS guidelines on prevention, investigation and management of post-operative endophthalmitis[END_REF] . This may explain our observation of visual outcome in the 4 patients with streptococcal endophthalmitis -there were 3 poor outcomes without vitrectomy and one good outcome after early vitrectomy. In this analysis, a link between visual outcome and socioeconomic deprivation was postulated. The CDS has been shown to correlate with prevalence of smoking, diabetes, obesity and vascular disease and may be regarded as a surrogate marker of systemic comorbidities [START_REF] Wild | Impact of deprivation on cardiovascular risk factors in people with diabetes: an observational series[END_REF] . Whilst no statistically significant relationship was identified in our small series, the concept of deprivation influencing visual outcome may benefit from further analysis in the context of a nationwide survey.

There are intrinsic limitations to a retrospective series such as this. The possibility that patients with endophthalmitis after surgery in our unit may have been missed on account of moving or seeking treatment elsewhere is unlikely but remains a theoretical limitation. Timing of presentation was not specifically defined for inclusion in this series. Despite this, there is a notable absence of late-onset cases. This limits our analysis to acute early-onset endophthalmitis only. Reliable statistical analysis is also precluded by small numbers. For instance, RAPD at presentation was not found to be a statistically significant predictor of visual outcome. However, there appears to be a trend towards significance and this important sign should be recorded in all cases.

In summary, the incidence of endophthalmitis after cataract surgery in this audit from a Scottish unit compares well with the current accepted UK benchmark [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . The microbiological profile of endophthalmitis also appears to be very similar to the rest of the UK with the majority of culture positive cases being caused by coagulase negative staphylococci [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF][START_REF] Mollan | Postcataract endophthalmitis: incidence and microbial isolates in a United Kingdom region from 1996 through 2004[END_REF] . Although visual recovery after endophthalmitis can be slow and variable, current treatment strategies will result in a significant proportion of patients regaining functional vision [START_REF] Kamalrajah | Surveillance of endophthalmitis following cataract surgery in the UK[END_REF] . Evidence-based approaches to prophylaxis of endophthalmitis will result in a significant reduction in its incidence over the next few years. The ESCRS multicenter study has shown a five-fold reduction from the routine use of intracameral cefuroxime at the end of cataract surgery [START_REF]ESCRS Endophthalmitis study group. Prophylaxis of postoperative endophthalmitis following cataract surgery: Results of the ESCRS multicentre study and indentification of risk factors[END_REF] . Published figures suggest that none of the 214 patients with endophthalmitis in the 1999 BOSU cohort had had intracameral antibiotics and, even in 2005, only 15% of eye departments in the UK were using this method of prophylaxis routinely [START_REF] Kamalarjah | Presumed infectious endophthalmitis following cataract surgery in the UK: a case-control study of risk factors[END_REF][START_REF] Gordon-Bennett | A survey of measures used for the prevention of postoperative endophthalmitis after cataract surgery in the United Kingdom[END_REF] . Whilst these figures seem to suggest a reluctance to introduce intracameral antibiotics, they predate the 2007 ESCRS findings [START_REF]ESCRS Endophthalmitis study group. Prophylaxis of postoperative endophthalmitis following cataract surgery: Results of the ESCRS multicentre study and indentification of risk factors[END_REF] . The ESCRS findings are poised to influence surgical practice in the UK and elsewhere and are already being replicated [START_REF] Yu-Wai-Man | Efficacy of intracameral and subconjunctiva cefuroxime in preventing endophthalmitis after cataract surgery[END_REF] . The importance of continuous surveillance and audit cannot be over-emphasized with respect to indentifying trends in post-operative endophthalmitis within individual eye units.

Table 1 -Patient characteristics (italics identify those with presumed low grade endophthalmitis)

 1

	Tap	BCVA

(M=male, F=female; RE= Right eye, LE=Left eye; Y= Yes, N=No; Growth + = light, ++ = moderare , +++=heavy; CF= counting fingers, HM= hand movements, PL= perception of light)

Table 3 -UK incidence of endophthalmitis

 3

	Authors				Year	Cohort	Incidence
	Desai et al 3		1997-1998	UK NCCS	0.03%*
	Kamalarajah et al	4	1999-2000	BOSU	0.14%
	Mayer et al	6		1991-2001	Taunton	0.16%
	Mollan et al	7		1997-2004	West-Midlands	0.099%
	Zaidi et al	8		2002-2004	London	0.1%
	This study			2000-2006	Glasgow	0.2%

(* incidence within 48hrs of surgery)

Table 4 -The microbiological profile of endophthalmitis from UK studies

 4

		This study	Kamalrajah et al	4	Mollan et al	7	Mayer et al	6
	Microbiological samples	88 % (22/25)	93% (199/213)		100% (105/105)	87% (26/30)
	Positive vitreous biopsy	60% (12/20)	53% (97/182)		53% (55/103)	N/A
	Positive AC tap	22% (4/18)	29% (36/126)		26%(26/101)	N/A
	Culture positive cases	55% (12/22)	56% (111/199)		58%(61/105)	52% (14/26)
	Gram positive isolates	100% (12/12)	93%(103/111)		93%(57/61)	76% (10/14)
	Coagulase negative staph	58% (7/12)	49%(54/111)		62%(38/61)	57% (8/14)
	Streptococcus sp	33% (4/12)	32%(36/111)		20%(12/61)	14% (2/14)

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Competing Interest

None declared


MD Ryo Asaoka

Nicholas G Strouthidis

Vasiliki Kappou

PhD Stuart K Gardiner

David F Garway-Heath
email: david.garway-heath@moorfields.nhs.uk

Strouthidis Md Ng

Mrcophth

V Kappou Bsc

M D Frcophth

HRT-3 Moorfields Reference Plane: Effect on Rim Area Repeatability and Identification of Progression

Keywords: HRT, Optic Nerve, Glaucoma, Visual Field

published or not. The documents may come   L'archive ouverte pluridisciplinaire

ABSTRACT:

Aims:

To assess the effect of the Moorfields Reference Plane on Heidelberg Retina Tomograph (HRT) rim area repeatability and its effect on progression rates using an event analysis.

Methods:

The HRT reference plane (RP) defines structures above as 'rim' and below as 'cup'. The Moorfields RP applies the Standard RP (located 50 μm posterior to the temporal disc margin) at baseline and maintains the distance between the Standard RP and the reference ring (located in the image periphery) for follow-up images.

The Moorfields RP was applied to an HRT test-retest dataset and rim area repeatability coefficients were calculated. Repeatability coefficients were compared between the Moorfields, Standard and 320 (located 320 μm posterior to the reference ring) RPs.

The Moorfields RP was applied to HRT images from 198 ocular hypertensives, acquired over 6 years. HRT progression required rim area baseline/follow-up differences exceeding the repeatability coefficient in two or more sectors, with confirmation in at least one of two consecutive images. Field progression was assessed using Advanced Glaucoma Intervention Study criteria.

Results:

The Moorfields RP improved rim area repeatability compared to the Standard RP; repeatability was similar between the Moorfields and the 320 RP. The frequency of identified progression using Moorfields RP was 40% compared to 28% for the 320 RP. There was a greater percentage with concurrent field progression -15.1% (Moorfields RP) compared to 12.1% (320 RP).

Conclusions:

Although rim area repeatability was similar using the 320 RP and the Moorfields RP, the latter resulted in greater rates of detection of change.

INTRODUCTION

Semi-automated optic nerve head (ONH) and retinal nerve fiber layer (RNFL) imaging devices have been commercially available for over a decade. These devices have potential applications in the management of patients with, or at risk of, glaucoma, both in assisting the clinician in the discrimination of normal optic discs from glaucomatous optic discs, [START_REF] Deleon-Ortega | Discrimination between glaucomatous and nonglaucomatous eyes using quantitative imaging devices and subjective optic nerve head assessment[END_REF][START_REF] Wollstein | Identification of early glaucoma cases with the scanning laser ophthalmoscope[END_REF][START_REF] Burgansky-Eliash | Glaucoma detection with the Heidelberg retina tomograph 3[END_REF][START_REF] Ferreras | Discriminating between Normal and Glaucoma-Damaged Eyes with the Heidelberg Retina Tomograph 3[END_REF] and in the monitoring of progression. [START_REF] Artes | Longitudinal changes in the visual field and optic disc in glaucoma[END_REF][START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF][START_REF] Kamal | Use of sequential Heidelberg retina tomograph images to identify changes at the optic disc in ocular hypertensive patients at risk of developing glaucoma[END_REF][START_REF] Kamal | Detection of optic disc change with the Heidelberg retina tomograph before confirmed visual field change in ocular hypertensives converting to early glaucoma[END_REF][START_REF] Strouthidis | Optic disc and visual field progression in ocular hypertensive subjects: detection rates, specificity, and agreement[END_REF][START_REF] Tan | Approach for identifying glaucomatous optic nerve progression by scanning laser tomography[END_REF] No consensus, however, yet exists as to how to best integrate these new technologies into clinical practice and what are the optimal operating criteria to enable their full potential to be realized.

The Heidelberg Retina Tomograph (HRT, Heidelberg Engineering, Heidelberg, Germany), which works on the principle of confocal scanning laser ophthalmoscopy, is the longest established of the scanning imaging devices used in glaucoma management. All new HRT software developments (the Explorer operational software is now in its third version) enable backward compatibility, so that images acquired using older versions of the device, such as the original HRT (now referred to as HRT 'Classic'), can be analyzed.

One approach to monitoring progression using the HRT is to monitor change in a particular stereometric parameter (such as rim area; RA) over time. A number of progression algorithms have been described which assess RA change. [START_REF] Artes | Longitudinal changes in the visual field and optic disc in glaucoma[END_REF][START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF][START_REF] Strouthidis | Optic disc and visual field progression in ocular hypertensive subjects: detection rates, specificity, and agreement[END_REF] RA has been proposed as a good marker for progression as it has been shown to be a repeatable, reliable and clinically meaningful HRT parameter. [START_REF] Strouthidis | Factors affecting the test-retest variability of Heidelberg retina tomograph and Heidelberg retina tomograph II measurements[END_REF][START_REF] Strouthidis | Improving the repeatability of Heidelberg retina tomograph and Heidelberg retina tomograph II rim area measurements[END_REF][START_REF] Tan | Variability across the optic nerve head in scanning laser tomography[END_REF] The greatest challenge in detecting progression is the ability to discriminate true change (disease process) from measurement variability. RA variability is strongly influenced by the position of the reference plane (RP). [START_REF] Strouthidis | Factors affecting the test-retest variability of Heidelberg retina tomograph and Heidelberg retina tomograph II measurements[END_REF][START_REF] Tan | Variability across the optic nerve head in scanning laser tomography[END_REF][START_REF] Breusegem | Variability of the Standard Reference Height and its Influence on the Stereometric Parameters of the Heidelberg Retina Tomograph 3[END_REF] The RP is parallel to, and below, the retinal surface and is used to define structures above the plane as 'rim' and below as 'cup'. The default RP in the Heidelberg Explorer software is the Standard RP (SRP), located 50 μm posterior to the temporal disc margin.

An alternative RP, the 320 RP is located 320 μm posterior to the reference ring (which is located in the image periphery) and has been shown to result in less RA variability. [START_REF] Strouthidis | Improving the repeatability of Heidelberg retina tomograph and Heidelberg retina tomograph II rim area measurements[END_REF] On this basis, our group has previously proposed the use of the 320 RP with RA-based progression algorithms. [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF][START_REF] Strouthidis | Optic disc and visual field progression in ocular hypertensive subjects: detection rates, specificity, and agreement[END_REF] We have recently described and tested a novel RP, the Moorfields RP (MRP). [START_REF] Poli | Analysis of HRT images: comparison of reference planes[END_REF] The MRP applies the Standard RP in the baseline image and then keeps the height difference between the baseline RP and the reference ring constant for follow-up images (Figure 1).

The aim of this study was to further investigate the performance of the MRP. Firstly, the effect of the MRP on RA repeatability was assessed. Secondly its effect on the detection of glaucoma progression was investigated by applying the MRP to an HRT RA event analysis (EA) algorithm.

MATERIALS AND METHODS

Assessment of Rim Area Repeatability:

Repeatability was assessed by applying the MRP to HRT images acquired as part of an HRT testretest study which has been described in detail elsewhere. [START_REF] Strouthidis | Factors affecting the test-retest variability of Heidelberg retina tomograph and Heidelberg retina tomograph II measurements[END_REF][START_REF] Strouthidis | Improving the repeatability of Heidelberg retina tomograph and Heidelberg retina tomograph II rim area measurements[END_REF] In summary, 43 eyes with ocular hypertension (OHT) and 31 with primary open angle glaucoma (POAG) were imaged by two experienced observers using the HRT Classic on the same date, and on a second date within 6 weeks of the date of the first image acquisition. Subjects had no previous history of intraocular surgery and had prior experience of ONH imaging using the HRT. The acquired HRT Classic single topographies were imported as HRTport files into a beta version of HRT Explorer Version 3.1.2.0, incorporating the MRP, with which mean topography images were generated.

The British Standards Institution definition of measurement error, the repeatability coefficient (RC), was used to quantify RA repeatability. [START_REF]Precision of Test Methods 1. Guide for the Determination and Reproducibility for a Standard Test Method[END_REF] 95% of inter-test differences are expected to lie within the value of the RC. RC was calculated as:

( ) ( ) ⎟ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎜ ⎝ ⎛ - = ∑ ns observatio n n observatio n observatio RC 2 2 1 * 2
Interobserver/intervisit RA RCs (using the MRP) were calculated for the 6 predefined HRT sectors -namely superotemporal (ST), inferotemporal (IT), temporal (T), superonasal (SN), inferonasal (IN) and nasal (N). HRT image quality was defined according to the mean pixel height standard deviation (MPHSD). Each subject eye was ranked by MPHSD (taken as the mean MPHSD of observation 1 and observation 2) with good quality images ranked in the upper tertile, medium quality in the middle tertile and poor quality images in the lower tertile. Sector RA RCs generated using the MRP were compared against previously published RCs generated using the SRP and 320 RP. [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF][START_REF] Strouthidis | Improving the repeatability of Heidelberg retina tomograph and Heidelberg retina tomograph II rim area measurements[END_REF] Assessment of HRT Progression:

The interobserver/intervisit RA RCs generated using the MRP were used as the basis for 'criteria for change' in an HRT event analysis progression algorithm. [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF] In the current study, HRT progression was flagged if a decrease in sector RA (baseline -follow-up) exceeded the sector RA RC for that level of image quality (taken as the mean of the baseline and follow-up MPHSD) in 2 or more sectors and confirmed in at least 2 out of 3 consecutive images. Significant improvement was defined using the same criteria, except requiring an increase in sector RA from baseline to follow-up. This strategy was classified as event analysis 2 (EA2) in our previous report. [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF] The EA2 strategy was applied to HRT mean topographies acquired using HRT 'Classic', from 198 OHT subjects and 21 control subjects followed prospectively with regular HRT and VF testing (1994 to 2001), originally recruited to a betaxolol versus placebo study. [START_REF] Kamal | Results of the betaxolol versus placebo treatment trial in ocular hypertension[END_REF] In brief, OHT was defined as an IOP > 22 mmHg and < 35 mmHg on 2 or more occasions within a 2-week period and a baseline mean Advanced Glaucoma Intervention Study (AGIS) VF score of 0 (Humphrey Field Analyzer, full-threshold 24-2 program). [START_REF] Agis | Advanced Glaucoma Intervention Study. 2. Visual field test scoring and reliability[END_REF] Control subjects were recruited from senior citizens groups or were the spouses or friends of subjects in the OHT cohort; they were not attending the eye clinic as patients and were not seeking care nor undergoing check-ups. [START_REF] Kamal | Detection of optic disc change with the Heidelberg retina tomograph before confirmed visual field change in ocular hypertensives converting to early glaucoma[END_REF] Controls had a baseline IOP of less than 21 mmHg, normal baseline VF test results (same criteria as in the OHT group), and were excluded if there was a self-reported family history of glaucoma or any coexistent ocular or neurological pathology. There was no significant age difference between the OHT and control groups (p = 0.09, student t-test) and both groups had similar ethnic backgrounds, both being predominantly (> 95%) of European descent. In the current study, the same eye was selected for analysis as had been randomised in the original study. The OHT eye randomisation was based on risk to glaucomatous conversion, classified according to pattern electroretinogram results, IOP, and cup-to-disc area ratio at the time of recruitment; [START_REF] Kamal | Results of the betaxolol versus placebo treatment trial in ocular hypertension[END_REF] control eyes were selected by simple randomisation.

HRT mean topographies were generated and analysed using a beta version of Heidelberg Explorer Version 3.1.2.0. (Heidelberg Engineering, Heidelberg, Germany). The MRP was used for all analyses. A single observer (NGS) drew contour lines onto the baseline mean topographies.

Contour lines were exported automatically to follow-up images. Manual alignment was used to correct for automatically 'misplaced' contour lines or if there was a magnification change. Each subject had a minimum of 5 HRT mean topographies, with all image qualities included except where satisfactory contour line alignment could not be achieved. In total, 8 mean topographies were excluded from the study, either as a result of 'double imaging' or if the image was so grainy as to prevent adequate visualisation of Elschnig's ring. The baseline HRT image was the first mean topography available for each subject and the baseline VF was taken as the VF test coinciding with, or nearest in acquisition date to, the baseline HRT mean topography.

In the absence of an accepted gold standard for defining glaucomatous progression, two proxy measures were used to generate an estimated 'specificity' for EA2 using the MRP. The first proxy measure was the number of control subjects (of 21) progressing and the second was the number of subjects (of 219) demonstrating significant improvement using the applied 'change' criteria.

The number of OHT subjects identified as progressing ('positive hit rate') was compared to the previously published results for the 320 RP applied to the same OHT cohort. [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF] HRT progression was compared to VF progression, which was identified if the AGIS VF score increased from 0 to 1 or more and was reproducible in three consecutive VF tests in the same region of the VF. [START_REF] Agis | Advanced Glaucoma Intervention Study. 2. Visual field test scoring and reliability[END_REF] Previous studies have estimated the specificity of the AGIS VF strategy at between 91 -100 %. [START_REF] Mayama | Statistical evaluation of the diagnostic accuracy of methods used to determine the progression of visual field defects in glaucoma[END_REF][START_REF] Zahari | Progression of visual field loss in open angle glaucoma in the Melbourne Visual Impairment Project[END_REF][21] The study adhered to the tenets of the Declaration of Helsinki and had local ethical committee approval, in addition to subjects' informed consent. All statistical analyses were performed using Medcalc Version 7.4.2.0 (Medcalc Software, Mariakerke, Belgium).

RESULTS

Interobserver/intervisit RA RCs for the SRP, 320 RP and MRP are shown in Table 1. Regardless of image quality, RA repeatability was generally poorer (higher RC values) for the SRP compared to the 320 RP and MRP. Differences were equivocal between RA repeatability using the 320 RP and the MRP, except in the temporal sector of medium and poor quality images where the MRP generated RCs of considerably greater magnitude than the 320 RP. A formal comparison of the intertest RA differences used to generate the RCs was performed using the generalized estimating equation technique. [START_REF] Zeger | Longitudinal data analysis for discrete and continuous outcomes[END_REF] This method is related to the standard least squares linear regression, but adjusts for the fact that the RA intertest differences from the six sectors are correlated as they are derived from the same subject (in each of the 74 data sets). Using the MRP, the mean intertest RA difference was 0.001 mm 2 greater than with the 320 RP, although this did not reach statistical significance (p = 0.229). Using the SRP, the mean intertest RA difference was 0.004 mm 2 greater than with the 320 RP (p = 0.04) and 0.003 mm 2 greater than with the MRP (p = 0.037).

The baseline demographics and characteristics of subjects included in the progression analysis are summarised in Table 2.

The specificity for the EA2 strategy following application of the MRP was estimated as follows: Application of the MRP to the EA2 strategy resulted in an increased number of OHT subjects identified as progressing by HRT compared to the previously published results for the 320 RP, 24.8% of OHT subjects compared to 16.2% (p = 0.046, Fisher's exact test). [START_REF] Fayers | Monitoring glaucomatous progression using a novel Heidelberg Retina Tomograph event analysis[END_REF] The agreement between progression identified by EA2 using either the MRP or the 320 RP and progression identified by AGIS criteria is illustrated in a Venn diagram (Figure 2). Of the 43 subjects who progressed according to the AGIS criteria, 70% also progressed by RA using the MRP, compared to 56% who progressed using the 320RP. This difference was not significant (p=0.265, Fisher's exact test).

DISCUSSION

The MRP was designed with the intention of combining positive attributes of both the SRP and the 320 RP, whilst at the same time compensating for their respective shortcomings. The 320 RP is anchored to a reference ring located in the image periphery. As such the height of the reference surface (the retina in the image periphery) is likely to be more stable than the reference surface for the SRP. The SRP is anchored at the temporal disc margin, a location which may shift posteriorly as glaucoma progresses and is subject to artifact in the surface height determination near Elschnig's ring. The SRP does, however, have an advantage over the 320 RP in that it can accommodate the large degree of morphological variation of the ONH, such as oblique insertion.

The 320 RP, by contrast, will be prone to generate inaccurate stereometric parameter measurements when applied to such ONHs.

The results of this study indicate that the 320 RP and MRP generate more repeatable RA measurements than the SRP, regardless of image quality. The differences in RA repeatability between the MRP and the 320 RP are equivocal. Our results suggest that the SRP has a greater tendency to generate 'noisy' RA measurements and should therefore not be considered for use when using RA to monitor progression. This does not, however, preclude the application of the SRP for other purposes; it is important to note that the Moorfields Regression Analysis algorithm uses RA measurements generated using the SRP. [START_REF] Wollstein | Identification of early glaucoma cases with the scanning laser ophthalmoscope[END_REF] Alternative progression algorithms, which are stereometric parameter-independent have been proposed. One such algorithm is topographical change analysis (TCA), which is incorporated into the current Explorer software. [START_REF] Chauhan | Technique for Detecting Serial Topographic Changes in Optic Disc and Peripapillary Retina Using Scanning Laser Tomography[END_REF] Our group has also described a method, statistic image mapping (SIM), which has been adapted from neuro-imaging techniques. [START_REF] Patterson | A new statistical approach for quantifying change in series of retinal and optic nerve head topography images[END_REF] Both TCA and SIM measure progression by monitoring pixel-based height change. Unlike RA, the anatomical correlates of SIM and TCA are less easily understood. HRT RA change has a direct anatomical corollorary, which is well understood and may be observed both by clinical examination and by assessment of optic disc stereo-photographs. It is possible that surface height changes within the disc margin, identified by either TCA or SIM, may reflect deformation at the level of the lamina cribrosa, but this has yet to be proven. [START_REF] Yang | 3-D Histomorphometry of the Normal and Early Glaucomatous Monkey Optic Nerve Head: Lamina Cribrosa and Peripapillary Scleral Position and Thickness[END_REF] TCA has been shown to have poor agreement as regards progression with disc stereo-photographs, [START_REF] Kourkoutas | Comparison of glaucoma progression evaluated with Heidelberg retina tomograph II versus optic nerve head stereophotographs[END_REF] The MRP identified more OHT subjects as progressing than the 320 RP, with a similar estimated specificity. The MRP showed improved agreement with VF progression, although this was not statistically significant. The estimated specificity range for EA2 using the 320 RP (94.1 -95.2%) is slightly higher than that generated for EA2 using the MRP (90.5 -93.2%); given the number of subjects included in these estimates it is likely that there is no real difference in specificity, however, small changes in specificity can have a large effect on the 'positive hit rate'. Given the similar values obtained for our two different approaches to obtain proxy measures of specificity, it is reasonable to postulate that the specificity of the criteria applied to the MRP-and the 320 RPgenerated RA are very similar. The comparison between progression rates using the different RPs is therefore fair and the observed differences in 'positive hit rate' are not simply the result of different specificity values.. These results suggest that, at a similar level of specificity, the MRP will identify more subjects as progressing than with the 320 RP and that these subjects are more likely to be genuine progressors. In our previous report we highlighted the higher 'signal to noise' ratio of the MRP and this most probably accounts for the improved detection of progression over the 320 RP, rather than RA repeatability which is similar between the two planes. Although these results require confirmation based on further prospective longitudinal studies, the apparent improved performance of the MRP over the 320 RP in detecting RA progression supports its clinical application in the longitudinal detection of structural change using HRT stereometric parameters

1 )

 1 Number of Subjects (OHT and Control) Improving = 15 (out of 219) Estimated Specificity = (204/219)*100 = 93.2% (95% confidence interval 88.7 -95.9 %) 2) Number of Controls Progressing = 2 (out of 21) Estimated Specificity = (19/21)*100 = 90.5% (95% confidence interval 68.2 -98.3%)

2 )Figure 1 .

 21 Figure 1. The location of the Moorfields Reference Plane. The height of the reference plane in the baseline image is the same as for the Standard Reference Plane, located 50 μm posterior to the temporal disc margin (white disc with black outline). The height difference between the baseline reference plane and the reference ring (black circle with grey outline) is kept constant for follow-up images.

Figure 2 .

 2 Figure 2. Venn diagram depicting the percentage of ocular hypertensive subjects (of 198) progressing by HRT Event Analysis 2 (EA2) and by AGIS visual field (VF) criteria. Values in bold are for the Moorfields Reference Plane and in italic are for the 320 Reference Plane.[6]. Estimated specificity for the HRT criteria is approximately 93%.

  whereas RA-based techniques have a slightly better agreement with disc photography (O' Leary N, et al. Invest Ophthalmol Vis Sci 2008;49:ARVO E-Abstract 5431).It should be stressed that stereo-photography is not an absolute gold standard for glaucomatous progression; therefore subjects identified as progressing by TCA or SIM but not by photographic examination may very well still be progressing, but in a fashion which cannot be appreciated using the latter technique. As most of our understanding regarding progressive structural changes in glaucoma has been based on careful clinical observations, we would encourage the continued use of stereometric parameter-based (particularly RA) progression algorithms in practice. This recommendation is not exclusive, however, as SIM and TCA are likely to pick up a proportion of genuine progressors which are different from those identified by RA change.

Table 1 . Interobserver/intervisit rim area repeatability coefficients (mm

 1

	T	N	ST	IT	SN	IN

ACKNOWLEDGEMENTS

The corresponding author has received a proportion of his funding from the Department of Health's National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health.

NGS is funded by a Royal College of Ophthalmologists/Pfizer fellowship and through an unrestricted grant from Heidelberg Engineering. DFGH has received research support from Heidelberg Engineering, OptoVue and Carl Zeiss Meditec and has acted as a consultant for Carl Zeiss Meditec.


MD Kati Kinnunen

MD Tuomo Puustjärvi

MD Markku Teräsvirta

MD Piia Nurmenniemi

Tommi Heikura

MSc Svetlana Laidinen

MD Timo Paavonen

MD Hannu Uusitalo

Tommi Heikura Msc

MD Seppo Ylä-Herttuala
email: seppo.ylaherttuala@uku.fi

Differences between type 1 and type 2 diabetics in retinal neovascular tissue and vitreous humour

Keywords: Growth factors, diabetic retinopathy, neovascularization

Introduction

The pathogenesis of diabetic retinopathy is characterized by alterations in retinal microvasculature. First sign of the diabetic retinopathy is a reduction of pericytes, leading to microvascular hyperpermeability, occlusion and hypoxia. When over 50% of the pericytes have been damaged, vascular cell proliferation begins. [START_REF] Enge | Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy[END_REF] Patients with retinal neovascularization have elevated levels of VEGF-A in their vitreous humour. [START_REF] Aiello | Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders[END_REF] Vascular endothelial growth factors (VEGFs) refer to a family of polypeptides with six members: VEGF-A, -B, -C, -D, placenta growth factor (PlGF) and a viral homologue VEGF-E. Their signalling is mediated via tyrosine kinase receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt-4). [START_REF] Ferrara | The Biology of Vascular Endothelial Growth Factor[END_REF] No information is available about other members of the VEGF family than VEGF-A in diabetic eyes. VEGF-B seems not to play an important role in retinal neovascularization. [START_REF] Reichelt | Vascular endothelial growth factor-B and retinal vascular development in the mouse[END_REF] VEGF-D is a close homologue to VEGF-C, produced as a prepropeptide and further processed to a biologically active form that binds to VEGFR-2 and -3 present on endothelial cells. [START_REF] Achen | Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)[END_REF][START_REF] Stacker | Biosynthesis of Vascular Endothelial Growth Factor-D Involves Proteolytic Processing Which Generates Non-covalent Homodimers[END_REF] VEGF-D induces angiogenesis and lymphangiogenesis. [START_REF] Marconcini | c-fos -induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro[END_REF][START_REF] Stacker | VEGF-D promotes the metastatic spread of tumor cells via the lymphatics[END_REF] Additionally, VEGF-D has been found in smooth muscle cells of arteries and arterioles. [START_REF] Achen | Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis[END_REF][START_REF] Rutanen | Vascular endothelial growth factor-D expression in human atherosclerotic lesions[END_REF] In the eye, the role of VEGF-D remains unclear. Similarly, the role of angiopoietin (ANG)-1 and ANG-2 remains unclear in diabetic retinopathy.

The aim of this study was to evaluate the presence of angiogenic factors in the proliferative neovascular tissues and vitreous humour of diabetic patients and to find out their possible roles in type 1 and type 2 diabetes.

Methods

Patient data

Informed consent was obtained from the subjects. The study protocol was approved by the Ethical Committee of Kuopio University Hospital and the study confirms with the principles outlined in the Declaration of Helsinki.

Vitreous sample of 13 type 1 diabetics and 17 type 2 diabetics was studied.

Additionally, neovascular tissue of 8 type 1 and 10 type 2 diabetic eyes was analyzed. Mean age of the patients in type 1 diabetics was 34.69±8.75 years (mean ± standard deviation (SD)) and in type 2 diabetics 60.41±8.43. The average duration of diabetes was 24.15±5.94 years in type 1 and 17.50±8.39 years in type 2 diabetics. Inclusion criterias were proliferative diabetic retinopathy and clinically observed retinal neovascularisation. Vitrectomy was indicated for all patients. All patients had had panretinal laserphotocoagulation before the surgical operation. Over all, the diabetic control was poor with the patients participating this study, average glucosylated haemoglobin (GHbA1c) being 10.16±1.9% in type 1 and 9.15±1.9% in type 2 diabetics. Exclusion criteria were previous vitreoretinal surgery, opening of the posterior lens capsule and less than 1 month old vitreous hemorrhage. Patient's age, gender, type of diabetes, duration of diabetes and the last value of GHbA1c were recorded. Vitreous control samples were collected from five patients undergoing pars plana vitrectomy and membranectomy due to idiopathic macular hole. Control tissue samples were analysed from the retinas of two non-diabetic normal eyes removed from trauma patients.

Vitreous body and neovascular samples

Vitreous humour samples were collected at the beginning of the pars plana vitrectomy into a 2 ml aspiration syringe. The sample was stored at -70 ○ C until analysed. A fibrovascular membrane was isolated and grasped with vitreous forceps to be pulled out from a sclerotomy. The sample was immediately covered with 4% PFA-sucrose, embedded in paraffin and processed for immunohistochemistry. [START_REF] Yla-Herttuala | Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions[END_REF]

Immunohistochemical analyses

Samples were immunostained with antibodies against endothelial cells (CD31, clone proliferating cell antigen (anti-Ki-67, clone 7B11, 1:100, Zymed Laboratories, Carlsbad, CA). [START_REF] Rissanen | VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses[END_REF] Controls for immunostainings included stainings were the first antibody was omitted and staining with species and class-matched irrelevant immunoglobulins. [START_REF] Yla-Herttuala | Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions[END_REF] Photographs of the histological sections were taken as described. [START_REF] Kinnunen | Overexpression of VEGF-A induces neovascularization and increased vascular leakage in rabbit eye after intravitreal adenoviral gene transfer[END_REF] ELISA assays VEGF-A, VEGF-D, ANG-1 and ANG-2 were analyzed with Enzyme-linked immunosorbent assay (ELISA) from vitreous samples as described. [START_REF] Kinnunen | Overexpression of VEGF-A induces neovascularization and increased vascular leakage in rabbit eye after intravitreal adenoviral gene transfer[END_REF]

Statistical Analyses

Statistical significances were evaluated using Kruskal-Wallis test followed by Mann-Whitney U-test where appropriate. Correlation analyses were performed using Spearman's correlation analysis. A value of P<0.05 was considered statistically significant.

Results

Type 1 diabetics

There were detectable amounts of VEGF-A, ANG-1 and ANG-2 in the vitreous samples but there was no difference between type 1 and type 2 diabetics (Figure 1). VEGF-D was not found from the vitreous samples. ANG-2 was found in low amounts also in the control eyes.

However, the difference between the control samples and the diabetic samples was significant (p<0.05). The amount of ANG-2 in the vitreous humour had a positive correlation with the number of endothelial cells (p<0.05) (Figure 2). The amount of VEGF-A in the vitreous humour had no correlation with other variables. GHbA1c levels did not correlate with the growth factor levels in the vitreous humour or neovascular samples.

In the neovascular samples, numerous CD31-positive endothelial cells and several CD68 positive macrophages were present (Figure 3). In addition, transcription factor NFκB was also present in the nuclei in the samples indicating an ongoing inflammatory process in the neovascular tissues (Figure 3). There were no NFκB positive cells in the control eyes. A positive staining for Ki-67 indicates a proliferative process in the tufts in all patients. Almost every sample had HIF-1α stained nuclei reflecting hypoxic conditions in the eye. There were no Ki-67 or HIF-1α positive cells in the control retinas (Figure 3). α-SMA positive pericytes were found in the neovascular tufts in the same areas as endothelial cells. There were more endothelial cells than activated pericytes in the tissues. Endothelial cells and α-SMA positive pericytes were also present in non-diabetic eyes (Figure 3). Number of endothelial cells and age had a significant negative correlation (p<0.05).

GHbA1c levels (p<0.01) had a positive correlation with the number of endothelial cells (Figure 4). VEGF-A was most strongly present in the samples. Also VEGF-D and ANG-2 were abundantly present (Table 1).

Overall order of the growth factors was VEGF-A > ANG-2 > VEGF-D, ANG-1 > VEGF-C > VEGF-B, PlGF > PDGF-B. ANG-1 and ANG-2 were mostly present in the same areas as pericytes.

Immunostaining for PDGF-B was weak (Figure 5). VEGFR-2 mediates most of the angiogenic effects of VEGFs in blood vessels.

Accordingly, several VEGFR-2 positive cells were found in the neovascular tufts. Strong stainings were also found for VEGFR-1 and VEGFR-3. The presence of VEGFR-1 and VEFGR-3 was significantly higher in type 1 diabetics than in type 2 diabetics (p<0.05). Tie-2 receptor was present in the samples and Tie-1 was found at the same level as Tie-2. Both of these receptors located in the same areas as ANG-1 and ANG-2 (Figure 5). VEGF-A and VEGFR-1 were also present in the control samples but much less abundantly than in the diabetic tissues.

Type 2 diabetics

The amounts of VEGF-A, VEGF-D, ANG-1 and ANG-2 in the vitreous samples did not differ between type 1 and type 2 diabetics in ELISA arrays (Figure 1). Unlike type 1 diabetics, amount of ANG-2 in the vitreous humour did not correlate with the number of endothelial cells.

VEGF-A and ANG-2 in the vitreous humour correlated positively (p<0.01) (Figure 2). GHbA1c levels, age or duration of diabetes did not correlate with the presence of VEGF-A in the vitreous humour. ANG-1 was found in the vitreous humour in much lower levels than other measured growth factors and it did not correlate with any other variables tested.

The number of endothelial cells in fibrovascular tufts was significantly lower than in type 1 diabetics (p<0.05). Despite of this, there were no differences in the presence of NFκB, CD68 positive macrophages, number of proliferating cells, immunostaining for HIF-1α or α-SMA stainings between type 1 and type 2 diabetics (Figure 3). Age of patient and duration of disease had a positive correlation (p<0.01) but neither had effect on the number of endothelial cells. Unlike in type 1 diabetics, GHbA1c levels did not correlate with the number of endothelial cells (Figure 4).

In type 1 diabetics, VEGF-A was most strongly present growth factor. Instead, VEGF-D was more abundantly present in the neovascular tissues of the type 2 diabetic patients than in those of the type 1 diabetic patients. Similarly also the amount of VEGF-B was increased in type 2 diabetics compared to type 1 diabetics in the neovascular tissues and the presence of VEGF-C was higher in type 1 diabetics than in type 2 diabetics (Table 1). All VEGF receptors were abundantly present but in lower levels than in type 1 diabetics. Presence and location of PDGF-B, ANG-1, ANG-2, Tie-1 and Tie-2 in type 2 diabetics were similar to type 1 diabetics (Figure 5). In type 2 diabetics, overall order of the growth factors was VEGF-D, VEGF-

A > ANG-2 > ANG-1 > PLGF > VEGF-B > VEGF-C > PDGF-B.

Discussion

Type 1 diabetics have much higher risk to develop proliferative retinopathy than type 2 diabetics [START_REF] Henricsson | Prevalence of diabetic retinopathy in relation to age at onset of the diabetes, treatment, duration and glycemic control[END_REF] and in type 2 diabetics, diabetic maculopathy is the most prevalent sight-threatening manifestation of retinopathy. [START_REF] Klein | The Wisconsin epidemiologic study of diabetic retinopathy. IV. Diabetic macular edema[END_REF] In this study, VEGF-A was most abundantly present in type 1 diabetics. However, VEGF-D was more abundantly present in the neovascular tissues of the type 2 diabetic patients than in type 1 diabetic patients. Both VEGF-A and VEGF-D angiogenic effects are mediated by VEGFR-2. Signalling of VEGF-A by VEGFR-2 is fast and transient whereas signalling of VEGF-D is slower and more long-lasting. [START_REF] Jia | Vascular Endothelial Growth Factor (VEGF)-D and VEGF-A Differentially Regulate KDR-mediated Signaling and Biological Function in Vascular Endothelial Cells[END_REF] In addition, Ikeda et al. have shown that in the human retinal pigment epithelium (RPE), cell-cell adhesion and attachment regulate the VEGF-D expression, suggesting that the breakdown of these interactions causes the overexpression of VEGF-D. [START_REF] Ikeda | The regulation of vascular endothelial growth factors (VEGF-A, -C, and -D) expression in the retinal pigment epithelium[END_REF] In the pathogenesis of vascular diseases in type 2 diabetes, the damage and dysfunction of endothelial cells have shown to play a crucial role. [START_REF] Tooke | Possible pathophysiological mechanisms for diabetic angiopathy in type 2 diabetes[END_REF] Similarly to studies in RPE cells, VEGF-D may contribute to the breakdown of the inner blood retina barrier in diabetic patients leading to maculopathy and oedema in type 2 diabetics. Macrophages secrete many angiogenic factors, including VEGF-D. [START_REF] Cursiefen | VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment[END_REF] Also in our patients, the cells with positive staining for VEGF-D, CD68 and CD31 were located in the same areas indicating that VEGF-D is produced by several different cell types in the neovascular tissues. Although the precise role of inflammation in the development of diabetic retinopathy is still unclear, it is possible that inflammatory mechanisms accelerate proliferative retinopathy in type 2 diabetics by VEGF-D dependent pathways. On the other hand, the fact that type 1 diabetics had more VEGF-A in the neovascular tissues than VEGF-D, suggests that with type 1 diabetics, hypoxia is more important in the development of proliferative retinopathy than inflammation. In order to stop the proliferative process, it might be beneficial also to prevent inflammation in the eye especially in type 2 diabetics. The difference in growth factor expression between type 1 and type 2 diabetic retinopathy might contribute to the pathogenesis of these diseases and also to differences in their clinical course. In both types of diabetics the immunoreactive VEGFR-1 and VEGFR-3 were abundantly present in the neovascular tufts. Expression of VEGFR-3 has also been found particularly in areas with leaky retinal microvessels in a previous study. [START_REF] Witmer | Altered Expression Patterns of VEGF Receptors in Human Diabetic Retina and in Experimental VEGF-Induced Retinopathy in Monkey[END_REF] This might suggest the important role of VEGFR-1 and VEGFR-3 in the formation and maintenance of microvessels such as retinal capillaries.

VEGF-A and ANG-2 were equally abundant in the vitreous humour. The amount of VEGF-A in the vitreous humour was at the same level as in previous studies, [START_REF] Aiello | Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders[END_REF] and also increased ANG-2 levels have been found in the vitreous humour of diabetic patients. [START_REF] Watanabe | Vitreous levels of angiopoietin 2 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy[END_REF] However, VEGF-D was not found in the vitreous humour in any patient. We cannot fully explain this phenomenon. However, it is known that VEGF-D binds to extracellular matrix components with high affinity. [START_REF] Stacker | Biosynthesis of Vascular Endothelial Growth Factor-D Involves Proteolytic Processing Which Generates Non-covalent Homodimers[END_REF] The finding that VEGF-D is absent in the vitreous humour suggests that VEGF-D binds to retinal tissues more efficiently than the other growth factors in diabetic patients.

HIF1-α has been found in the epiretinal membranes of diabetic patients without previous laser photocoagulation, [START_REF] El-Asrar | Expression of hypoxia-inducible factor-1{alpha} and the protein products of its target genes in diabetic fibrovascular epiretinal membranes[END_REF] and it was also present in this study despite of the previously performed panretinal laser photocoagulation. Although the retinal vessels are autoregulated in hypoxic conditions, it seems that in diabetic retinopathy, the normal attenuation mechanism of the eye fails leading to active angiogenic process and proliferation.

In conclusion, there were differencies in the measured angiogenic factors between type 1 and type 2 diabetic retinopathy. In type 2 diabetics, VEGF-D was more abundantly present than in those of the type 1 diabetics. Also, ANG-2 seems to be an important factor in neovessel growth in both types of diabetes. These differencies should be considered when developing new treatment options for patients with proliferative diabetic retinopathy. In type 1 diabetics, it is most likely that blocking VEGF-A, VEGFR-2 and ANG-2 can cause therapeutic effects but in type 2 diabetics, also preventing the action of VEGF-D and other VEGFRs could be beneficial. The tissues are also hypoxic (HIF1-α (I)). There are few active pericytes (α-SMA (K)) present in the diabetic retinas but none in the control retinas (L). There are no CD68 or NFκ-B positive cells in the control retinas (D,F). Furthermore, proliferative process or hypoxia is absent in the control tissues (H,J). Brown colour indicates positive staining. Scale bar = 0.3mm (applies to all panels). Figure 4. Scatter Plot analysis of type 1 and type 2 diabetic patients with proliferative retinopathy. A) Correlation between duration of diabetes and the age of patients (p<0.05). B) Correlation between the number of CD-31 positive endothelial cells in the tissue/mm 2 and age of patients (p<0.05). C) Correlation between the number of CD-31 positive endothelial cells in the tissue/mm 2 and glucosylated haemoglobin (GHbA1c, %)(p<0.01). P<0.05 is considered statistically significant. Figure 5. Growth factors and their receptors in the neovascular tissues of diabetic patients with proliferative retinopathy. Expression of vascular endothelial growth factor (VEGF) -A is strong (A,B) in both types of diabetes. VEGF-B is more abundant in type 2 diabetes (C,D) and VEGF-C in type 1 diabetes (E,F). In type 2 diabetics, the expression of VEGF-D (G,H) is more abundant than in type 1 diabetics. Angiopoietin (ANG) -1 is equally abundant (I,J) and there was a strong staining for ANG-2 (K,L) in both types of diabetes. Placenta derived growth factor (PlGF) (M,N) and platelet derived growth factor (PDGF-B) (O,P) were less abundant in the tissues. Both Tie-1 (Q,R) and Tie-2 (S,T) were equally abundant in the tissues. The presence of VEGF receptor 1 (VEGFR-1)(Flt-1;U,V) was significantly higher in type 1 than in type 2 diabetics. In addition to VEGF receptor 2 (VEGFR-2)(KDR;W,X), also VEGFR-1 and VEGF receptor 3 (Flt-4;Y,Z) were abundantly present in the neovascular tissues. Brown colour indicates positive staining. Haematoxyline-eosin stained control sample (Å). Scale bar = 0.3mm (applies to all panels).

JC70A, 1 : 1 (

 11 50, Dako, Carpinteria, CA) macrophages (CD68, clone KP1, 1:50, Dako, Carpinteria, CA), smooth muscle cells (α-SMA, clone 1A4, 1:200, Sigma, St Louis, MO), VEGF (VEGF-A, clone sc-7269, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), VEGF-B (VEGF-B, clone sc-1878, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), VEGF-C (VEGF-C, clone sc-1881, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), VEGF-D (VEGF-D, clone 78923.11, 1:500, R&D Systems, Minneapolis, MN), placenta growth factor (PlGF, clone sc-1880, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA ), platelet derived factor B (PDGF-B, 1:100, R&D Systems, Minneapolis, MN), ANG-1 (ANG-1, clone sc-6319, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), ANG-2 (ANG-2, clone sc-7015, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), Tie-Tie-1, clone 88016, 1:25, R&D Systems, Minneapolis, MN), Tie-2 (Tie-2, clone 83715, 1:25, R&D Systems, Minneapolis, MN), hypoxia inducible factor (HIF-1α, clone H1α67, 1:100, Neomarkers, Fremont, CA), vascular endothelial growth factor receptors -1 (Flt-1, clone sc-316, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), -2 (KDR, clone sc-6251, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and -3 (Flt-4, clone sc-321, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), nuclear factor κ B (NF-κB, clone 20, 1:250, BD Biosciences, San Jose, CA) and

Figure 1 .

 1 Figure1. Elisa analyses of vitreous humour in type 1 diabetic (n=13), type 2 diabetics (n=17) and controls (n=5). VEGF-A, VEGF-D, ANG-2 and ANG-1 were analysed. P<0.05 is considered statistically significant.

Figure 2 .

 2 Figure 2. Scatter Plot analysis of type 1 and type 2 diabetic patients with proliferative retinopathy. A) Correlation between the number of CD-31 positive endothelial cells in the tissue/mm 2 and ANG-2 in vitreous humour (p<0.05). B) Correlation between ANG-2 and VEGF-A in vitreous humour (p<0.01). P<0.05 is considered statistically significant.

Figure 3 .

 3 Figure 3. Neovascular tissues of diabetic patients with proliferative retinopathy and the retina of control patients. CD31 (A,B) shows lots of endothelial cells in the samples. Inflammatory response is strong in the diabetic tissues (macrophages; CD68 (C) and NFκ-B; (E)). Ki67 indicates active proliferation in the diabetic tissues (G). The tissues are also hypoxic (HIF1-α (I)). There are few active pericytes (α-SMA (K)) present in the diabetic retinas but none in the control retinas (L). There are no CD68 or NFκ-B positive cells in the control retinas (D,F). Furthermore, proliferative process or hypoxia is absent in the control tissues (H,J). Brown colour indicates positive staining. Scale bar = 0.3mm (applies to all panels).

Acknowledgments

The authors want to thank Helvi Käsnänen, Anne Kaakkola and Seija Sahrio for skilful technical assistance. This manuscript was supported by grants from Finnish Academy, Finnish Diabetes Research Foundation, Finnish Cultural Foundation, Kuopio University Hospital (EVO Grant 5187), Evald and Hilda Nissi Foundation and Finnish Eye Foundation.

Authors have no financial or proprietary interest in products mentioned in the article.

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Competing Interest: None declared.