Heather Thomson

Andrew Treharne

Paul Walker

Martin Grossel

Andrew Lotery
email: a.j.lotery@soton.ac.uk

Prof Andrew Lotery

Optimisation of polymer scaffolds for RPE cell transplantation

Keywords: Poly(α-hydroxy esters), Polymer, Biodegrabable, Scaffold, Retinal Pigment Epithelium

Aim: To evaluate a variety of copolymers as suitable scaffolds to facilitate retinal pigment epithelium (RPE) transplantation.

Methods: Five blends of poly(L-lactic acid) (PLLA) with poly(D, L-lactic-glycolic acid) (PLGA) were manufactured by a solid-liquid phase separation technique. The blends were 10:90, 25:75, 50:50, 75:25, 90:10 (PLLA:PLGA). All blend ratios were validated by nuclear magnetic resonance spectroscopy. Samples of polymer blends were coated with laminin. Coated and uncoated blends were seeded with a human RPE cell line. Cell attachment, viability and retention of phenotype were assessed.

Results: As the lactide unit content increased pore size generally became smaller. The 25:75 PLLA:PLGA blend was the most porous (44%) and thinnest (134μm) scaffold produced. ARPE-19 cells survived with minimal cell death and maintained their normal phenotype for up to four weeks.. Cell density was maintained with only one of the fabricated ratios (25% PLLA:75% PLGA). There was a consistent decrease in apoptotic cell death with time on laminin coated samples of this blend. A decrease in polymer thickness concomitant with an increase in porosity characteristic of degradation was observed with all polymer blends.

Conclusions:

This study demonstrates that a 25:75 copolymer blend of PLLA:PLGA is a potentially useful scaffold for ocular cell transplantation.

Introduction

Biomaterial transplants offer a means of restoring function in biological structures which cannot otherwise be repaired [START_REF] Barbanti | Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture[END_REF] . One such ocular structure is the retinal pigment epithelium (RPE). RPE degeneration is fundamental to the progression of common causes of blindness such as age-related macular degeneration (AMD) [START_REF] Lotery | Progress in defining the molecular biology of age related macular degeneration[END_REF] . Therefore RPE transplantation would be a novel treatment.

The principal challenges involved in cell transplantation include delivery, localisation and survival of cells. Cell delivery to the retina is typically achieved by injection of cell suspensions into the sub-retinal space. However there are a number of drawbacks associated with this method including cell death, reflux from the injection site and incorrectly localised or disorganised grafts 3;4 .

Interestingly recent studies have demonstrated that growth of RPE and retinal progenitor cells on polymer scaffolds can facilitate transplantation of cells as an intact functioning monolayer which should overcome these undesirable sequela [START_REF] Lavik | Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors[END_REF] . Delivery of cells as composite polymer grafts reduced cell death and improved overall survival by up to 10 fold [START_REF] Tomita | Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells[END_REF] . However there have only been limited studies to identify the optimal polymer composition for such grafts [START_REF] Lavik | Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors[END_REF][START_REF] Tomita | Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells[END_REF][START_REF] Lu | In vitro and in vivo degradation of porous poly(DL-lacticco-glycolic acid) foams[END_REF][START_REF] Ng | Creating an immune-privileged site using retinal progenitor cells and biodegradable polymers[END_REF] .

Polymers are macromolecules composed of many subunits which form linear, branched, or crosslinked structures [START_REF] Hasirci | Versatility of biodegradable biopolymers: degradability and an in vivo application[END_REF] . The poly(α-hydroxy esters) have been extensively investigated for tissue engineering applications. They allow controlled degradation and facilitate the attachment of anchorage-dependent cells, which require a supportive matrix for survival and growth [START_REF] Lu | Retinal pigment epithelium engineering using synthetic biodegradable polymers[END_REF] . Additional benefits of these polymers include good biocompatibility with the possibility of processing into desired configurations of controlled thickness [START_REF] Lu | Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films[END_REF] . Two promising polymers for consideration as ocular bio-scaffolds are poly(L-lactic acid) (PLLA) and poly(D,L-lactic-glycolic acid) (PLGA).

They are biocompatible within the eye [START_REF] Sakurai | Scleral plug of biodegradable polymers containing tacrolimus (FK506) for experimental uveitis[END_REF] , and have been successfully employed for various biomedical applications including gene, growth factor and drug carriage [START_REF] Guosen | Design, synthesis and in vitro evaluation of a novel "stealth" polymeric gene vector[END_REF][START_REF] Dong | d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified poly(l-lactide) (PLLA) films for localized delivery of paclitaxel[END_REF][START_REF] El-Ayoubi | Design and fabrication of 3D porous scaffolds to facilitate cell-based gene therapy[END_REF] . Both have been utilised for culture and transplantation of retinal cells 5;6;15;16 . Previous studies have evaluated a (50 PLLA: 50 PLGA) copolymer blend for ocular cell attachment 5;6 .

The purpose of the present study was to fabricate a range of novel polymer blends and to evaluate their suitability as a bio-scaffold. An RPE cell line was seeded onto samples of each polymer blend and maintained in vitro for up to 4 weeks.

Materials and Methods

Poly(L-lactic acid) (PLLA) Resomer® L 207 S, inherent viscosity (i.v.) 1.5-2.0dL/g and poly(DLlactide-co-glycolide) (PLGA) Resomer® RG 755 S, i.v. 0.5-0.7dL/g, with a lactic to glycolic ratio 75:25 were obtained from Boehringer Ingelheim GmbH (Ingelheim, Germany). All solvents were obtained from Fisher Scientific (Loughborough, UK). Polymers scaffolds were prepared as previously described [START_REF] Lavik | Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors[END_REF] . Five blends of PLLA with PLGA were prepared in neat 1,4-dioxane (Fig 1B). 0.5ml of each copolymer solution was applied to the surface of an uncoated microscope slides and allowed to spread uniformly. Slides were then placed on a metal grid sitting on ice for 1 minute. A dry-ice cooled copper wire was touched to the surface of the solution to initiate solidification. Slides remained on ice for a further minute to solidify the dioxane before being transferred to a -20 o C freezer overnight. Prior to use, polymers were placed under high vacuum at room temperature to completely remove dioxane.

Inverse-gated [START_REF] Dong | d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified poly(l-lactide) (PLLA) films for localized delivery of paclitaxel[END_REF] C NMR spectra were obtained for samples of all copolymer blends using established methods [START_REF] Hausberger | Characterization of biodegradable poly(D,L-lactide-coglycolide) polymers and microspheres[END_REF] . CDCl 3 was used as the solvent for all samples, polymer concentration used was 0.02g/ml. δ( Cell seeded and unseeded polymer samples maintained in culture for up to one month were fixed in 3% glutaraldehyde with 4% formaldehyde in 0.1M Piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES) buffer (pH 7.2), followed by dehydration through a series of graded ethanol steps to 100% ethanol. Samples were then critical-point dried with CO 2 using a critical point drier (Balzers, Liechtenstein) prior to mounting on stubs and sputter-coating with gold/palladium. SEM was performed on a Quanta 200 (FEI, Eindhoven, Netherlands) scanning electron microscope.

Micrographs of four random areas of each scaffold were taken.

A RPE cell line (ARPE-19) was purchased from the American Tissue Culture Collection (ATCC) (Manassas, VA, USA). Cells were maintained at 37 o C in a humidified atmosphere with 5% CO 2 in culture medium comprising 1:1 (vol/vol) mixture of D-MEM:F12 (ATCC) supplemented with 1% antibiotic-antimycotic solution (10,000 units/ml penicillin G, 10 mg/ml streptomycin sulphate, [START_REF] Ronneberger | In vivo biocompatibility study of ABA triblock copolymers consisting of poly(L-lactic-co-glycolic acid) A blocks attached to central poly(oxyethylene) B blocks[END_REF] μg/ml amphotericin B) (Sigma-Aldrich, Ayrshire, UK) and 10% fetal bovine serum (FBS) (ATCC).

Scaffolds were cut into 10mm 2 sections and sterilised overnight in 70% ethanol. Under aseptic conditions scaffolds were rinsed 3 times in phosphate buffered saline (PBS) and then transferred to 6-well plates (Fisher Scientific). Half of the polymers prepared for each blend were coated with 1ml Laminin (0.5μg/ml) (Invitrogen, Paisley, UK) for 60 minutes at 37 o C, the other half remained uncoated. Scaffolds were rinsed 3 times with PBS, all samples were then flooded with culture medium and incubated for 30 minutes at 37 o C. Cells were seeded at a density of 4x10 4 per 100μl.

Cells were allowed to attach for 30 minutes, 4mls of culture medium was then added to each well.

Cell seeded polymers were maintained for 4 days, polymer/cell constructs were then seeded at the same density on the opposite side of the polymer. Culture medium was changed every 48 hours, samples of polymer were taken 1, 2 and 4 weeks post-seeding.

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was used to detect DNA fragmentation resulting from apoptosis. Samples of seeded polymer were cryosectioned (15μm) and fixed in 4 % paraformaldehyde (PFA) 1, 2 and 4 weeks post-seeding.

Evaluation of apoptotic cell death was performed using a commercially available kit (Promega, Madison, WI, USA) in accordance with manufacturers' instructions. DNase I treatment was used as a positive control and exclusion of rTNT from the reaction buffer was used as the negative control.

Cell nuclei were visualised with propidium iodide (PI) (Invitrogen). PI positive and TUNEL positive cell areas were evaluated using the density slice function on Improvision Volocity software (Improvision, Coventry Lexington, UK). At 1, 2 and 4 weeks scaffolds were cryosectioned at 15μm and fixed in 4% PFA. Sections were stained with antisera directed against Proliferating Cellular Nuclear Antigen (PCNA) (1:6000 Sigma-Aldrich), Cytokeratin 18 (1:250 Abcam, Cambridge, UK), Cellular Retinaldehyde-Binding Protein (CRALBP) (1:250 Abcam) and Retinal Pigment Epithelium-specific protein 65kDa (RPE65) (1:250 Abcam). Samples were incubated overnight at 4 o C with primary antibody. Samples were then incubated for 2 hours at room temperature with an Alexa Fluor 488-conjugate (1:1000 Invitrogen). Cell nuclei were visualised with 4',6'-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Samples were imaged using a Leica DM IRB microscope (Leica Microsystems UK Ltd, Milton Keynes, UK). Cryosections were also used to determine cell area by nuclear labelling with PI, polymer thickness and porosity. Ten fields were imaged per condition at each time point. Cell area was measured in relation to total polymer area, with analysis carried out using Volocity software (Improvision).

All data were analyzed by either Student's t-test or one way analysis of variance (ANOVA) using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA, USA). Post-hoc comparisons were made using the Tukey-Kramer Multiple Comparisons Test when the p-value was significant (p<0.05).

Results

Scaffolds were fabricated using a solid-liquid phase separation technique which led to the formation of a reproducible pore structure, which was a direct result of the freezing process. As the lactide unit content was increased through the blends, pores generally became smaller. The 10:90

blend gave an open macroporous structure with pore size ranging from 20-70μm (Fig. 1A(i)).

While the 90:10 blend had surface pores (10-20μm) with smaller micropores below the surface, approximately ten fold smaller (Fig. 1A(v)). Surface porosity was quantified using the density slice function of Volocity software (Improvision). Average porosity prior to seeding was 33.2% ± 2.3, the 25:75 PLLA:PLGA blend was the most porous scaffold with an average porosity of 44.4% ± 0.1, while the 50:50 blend was the least porous (25.4% ± 0.2). Polymer thickness was determined using Volocity software. The 50 PLLA:50 PLGA polymer blend was the thickest produced while the thinnest was the 25:75 blend. Average overall thickness at week 1, 2 and 4 post seeding was 186.3 μm ± 3.7, 172.7 μm ± 3.4 and 143.9 μm ± 3.9 respectively (Table 1). The polymers used in this paper are commercially available, however it was important to validate the blends produced. Inverse-gated [START_REF] Dong | d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified poly(l-lactide) (PLLA) films for localized delivery of paclitaxel[END_REF] C NMR was used to determine the ratio of lactide to glycolide units within the copolymer blends. Initially an inverse-gated [START_REF] Dong | d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified poly(l-lactide) (PLLA) films for localized delivery of paclitaxel[END_REF] C spectrum was obtained for PLGA. This spectrum was used to check the veracity of the lactide to glycolide units composition supplied by the manufacturer. The spectrum showed close agreement to the anticipated 75:25 ratio. Most spectra showed the presence of slightly more lactide units than expected (Table 2). For the 90:10 PLLA:PLGA blend, a peak for the methylene group in the glycolide unit was not observed above spectral noise due to the relatively small percentage of glycolide units present in this blend (2.5% predicted).

Table 2 The pore architecture of the manufactured polymer blends allowed for cell attachment which is important for proliferation and maintenance of function for anchorage dependent cells [START_REF] Giordano | Retinal pigment epithelium cells cultured on synthetic biodegradable polymers[END_REF] . Cells were noted on all polymer blends and were also seen to populate the interior of the scaffolds as visualised by transmission electron microscopy (data not shown). Laminin coating did not appear to affect cell attachment.

There was significantly less cell death (p<0.001) at all time points when compared to positive control (cells treated with DNase I) (Fig. 2 A;B). Minimal cell death was noted 1, 2 and 4 weeks post-seeding, average overall cell death was 6.6% ± 0.8, 9.1% ± 0.8 and 10.4% ± 0.9 respectively (Fig. 2C). Average overall cell death on uncoated polymer blends was 9.7% ± 0.8, while on laminin coated polymers cell death was 9.2% ± 0.5, this difference was not significant (p=0.597) (Fig. 2D). Cell death was however reduced at all time points on the laminin coated 50:50 polymer blend. This effect did not reach significance. Cell death consistently decreased over the investigation period on the coated 25:75 polymer blend only. Cell death at week 1 (11.0% ± 2.6) decreased to 5.4% ± 1.5 by week 4 (p= 0.056) (Supplementary Table 1).

Quantification of cell density revealed that cells covered an average of 37.5%, 38.2% and 36.5% of the total polymer area at 1, 2 and 4 weeks post seeding. Average porosity was quantified as 35.2% ± 2.1 (week 1), 40.8% ± 2.1 (week 2) and 46.8% ± 1.6 (week 4) (Table 1). Only the 25:75 ratio maintained ARPE-19 cell proliferation/attachment throughout the investigation period.

Morphology of ARPE-19 cells was unaffected by polymer ratio when compared to cells grown on tissue culture plastic. Scanning electron micrographs taken 7 days post seeding revealed numerous apical microvilli present on ARPE-19 cells (Fig. 1C, Supplementary Fig. 1). ARPE-19 cells also retained a characteristic cuboidal morphology with centrally located nuclei on all polymer blends.

Staining was carried out for the non-squamous epithelial cell marker cytokeratin 18 and characteristic phenotypic RPE cell markers (RPE65 and CRALBP) (Fig. 3, Supplementary Fig. 2).

Gradual hydrolysis of the polymer component was seen over the investigation period. There was an average decrease in polymer thickness of 23.7% ± 5.7. Variations in pore size within the different polymer blends did not significantly affect the degradation process 1 . However degradation was observed as an increase in pore size. Large channels were noted 2 and 4 weeks post seeding in all polymers. Cross sectional porosity was quantified as a percentage of total polymer area ((Fig. 1D (i-iii) 42.9 % ± 3.8, 45.1% ± 5.8 and 64.9% ± 2.5). Laminin coating did not affect the rate of degradation of the polymer blends studied.

Discussion

In vitro models offer a controlled environment in which the direct effect of polymer composition on cell adhesion, proliferation, and viability can be investigated [START_REF] Huhtala | In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: direct contact studies[END_REF] . In the present study we addressed the questions of cell attachment, proliferation and maintenance of phenotypic characteristics in a range of copolymer blends of PLLA:PLGA. A well-characterized human RPE cell line (ARPE-19) was used to investigate cell attachment and potential toxicity in blends of PLLA:PLGA (10:90-90:10). We verified the copolymer blend ratios by inverse-gated NMR Spectra produced by this method were in good agreement with those predicted.

Primarily we investigated cell adherence to the polymer blends. Samples of polymer/cell constructs were examined 1, 2 and 4 weeks post seeding. Microscopic analysis showed no obvious differences in cellular morphology between the polymer blends. Cells readily adhered to and proliferated on all materials tested. Minimal cell death was noted over the investigation period. Coating with laminin, an ECM component which plays a crucial role in cell migration and differentiation [START_REF] Huang | Surface modification and characterization of chitosan or PLGA membrane with laminin by chemical and oxygen plasma treatment for neural regeneration[END_REF] , did not alter cell attachment or survival. Chemical bonding of laminin or fragments of the laminin protein may produce a more significant effect [START_REF] Hersel | RGD modified polymers: biomaterials for stimulated cell adhesion and beyond[END_REF] . ARPE-19 cells may transdifferentiate to a fibroblast-like state in vitro [START_REF] Opas | Substratum mechanics and cell differentiation[END_REF] . However in this study analysis using characteristic RPE cell markers, demonstrated retention of phenotype. Importantly, for cell transplantation to the sub-retinal space, ultra-structural inspection of cellular morphology with SEM showed monolayers of ARPE-19 cells with no apparent multilayering. Numerous apical microvilli were present on the surface of ARPE-19 cells.

Microvilli normally interdigitate with the outer segments of photoreceptors and mediate phagocytosis of rod outer segments [START_REF] Lee | Spatial cues for the enhancement of retinal pigment epithelial cell function in potential transplants[END_REF] , thus the presence of microvilli on the surface of cultured ARPE-19 cells is indicative that these cells had retained functional integrity in vitro.

Polymer environment, monomer ratio, and scaffold thickness have a major influence on the kinetics of degradation [START_REF] Spenlehauer | In vitro and in vivo degradation of poly(D,L lactide/glycolide) type microspheres made by solvent evaporation method[END_REF] . Degradation rate is governed by the ratio of hydrophilic polyglycolic acid to hydrophobic polylactic acid [START_REF] Lu | In vitro and in vivo degradation of porous poly(DL-lacticco-glycolic acid) foams[END_REF] . A high degree of hydrophilicity facilitates the absorption and diffusion of water leading to hydrolysis, accordingly the 10:90 and 25:75 PLLA:PLGA polymer blends appeared to have degraded more when compared to the other more lactide rich blends.

Degradation time is fundamental for successful biocompatibility and functionality [START_REF] Barbanti | Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture[END_REF] . It is important for polymers to act as a substrate for a sufficient time for cells to become confluent and establish essential cell-cell interactions. Excessively long degradation times may however induce chronic inflammatory reactions in vivo [START_REF] Ronneberger | In vivo biocompatibility study of ABA triblock copolymers consisting of poly(L-lactic-co-glycolic acid) A blocks attached to central poly(oxyethylene) B blocks[END_REF] . PLLA alone has a degradation time of 1 to 2 years while PLGA may degrade within weeks. Thus neither polymer alone would be ideal for transplantation. This prompted our evaluation of these polymers as composite copolymers.

Only the 25:75 (PLLA:PLGA) ratio blend enabled maintenance of cell proliferation and attachment throughout the study. This blend was the thinnest produced, and also the most porous. The balance between lactide units and thickness ensured that the polymer scaffolds did not fully degrade over the investigation period thus affording structural support to cells throughout. These findings are indicative that for RPE cell growth the 25:75 (PLLA:PLGA) ratio produced is the most efficacious.

Preliminary work such as this is required prior to assessment in animal models of disease. Such research is necessary to develop innovative treatments for common causes of blindness such as age related macular degeneration.

Table 1

 1

		Thickness	SEM	Porosity	SEM	Total Area	SEM	Cell area	SEM
		(μm 2 )		(%)		(μm 2 )		(μm 2 )
	10:90
	week 1	196.8	4.1	39.7	3.3	67617	2552	23939	1835
	week 2	170.5	4.9	45.7	0.7	52843	2597	25138	1362
	week 4	107.3	1.9	55.0	2.8	47820	1500	16463	1284
	25:75
	week 1	133.1	3.5	42.9	3.8	56634	2979	33337	3706
	week 2	131.5	2.3	45.1	5.8	51423	1577	22149	1104
	week 4	103.5	1.2	64.9	2.5	40546	2758	40304	2513
	50:50
	week 1	229.1	3.6	26.2	2.2	110885	25042	10477	938
	week 2	200.4	1.5	34.0	3.3	100264	12897	28041	3029
	week 4	183.9	8.8	37.9	1.2	70951	9061	17913	887
	75:25
	week 1	197.2	4.3	32.6	1.8	70234	852	17543	1599
	week 2	190.3	12.4	37.4	7.8	68119	3014	27923	2103
	week 4	160.6	4.3	38.4	1.9	66783	9649	17265	1045
	90:10
	week 1	177.9	10.2	34.7	7.2	63533	1057	15975	2169
	week 2	177.0	2.7	38.2	1.8	60265	6517	24168	1288
	week 4	155.5	3.5	49.1	3.0	58611	1100	11955	784

Acknowledgements

-We thank the Biomedical Imaging Unit, School of Medicine and the Science and Engineering Electron Microscopy Centre, School of Chemistry, University of Southampton for assistance with SEM. We would also like to thank the School of Chemistry NMR facility. Funding-Financial support was provided by Foresight RP, the Gift of Sight Appeal, Lord Sandberg and the Biotechnology and Biological Sciences Research Council.

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://group.bmj.com/products/journals/instructions-for-authors/licence-forms).

Competing Interests-None

Legends to Figures

 B,E,H,K,N). Merged images (C, F, I, L, O).

Scale bar-13μm

Table 1 Table depicting average scaffold thickness, porosity, polymer area for cell attachment and cell area at 1, 2 and 4 weeks post seeding with ARPE-19 cells. Ten fields were imaged per condition at each time point. Porosity and cell area were measured in relation to total polymer area.

Measurements and analysis carried out using Volocity software (Improvision). Results are mean ± SEM (n=10) per condition.

Table 2 [START_REF] Dong | d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified poly(l-lactide) (PLLA) films for localized delivery of paclitaxel[END_REF] C Nuclear Magnetic Resonance Spectroscopy ratios for each polymer blend. Table

showing NMR predicted and spectral ratios for each polymer blend, with lactide and glycolide unit calculations for the predicted (in bold) and spectral values.

Supplementary Table 1-Table comparing ARPE


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

Nicholas Leon

Shehzad Anjam Davies

Phillip Jonathan Naroo

George Anthony Buckhurst

Navneet Gibson

Jennifer Patricia Gupta

Sunil Craig

Shah

Leon Nicholas Davies

Shehzad Anjam Naroo

Phillip Jonathan Buckhurst

George Anthony Gibson

Navneet Gupta

Jennifer Patricia Craig

Sunil Shah

Evaluation of an open-field autorefractor's ability to measure refraction and hence potential to assess objective accommodation in pseudophakes

Keywords: intraocular lens, pseudophakia, objective accommodation, autorefractor

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Objective measurement of the myopic shift that occurs with the effort to focus at near due to alterations in crystalline lens surface curvatures or intraocular lens position, refractive indices or surface curvatures, has become of increased interest to better understand accommodation and attempts to develop 'accommodating' IOLs. Subjective amplitude-ofaccommodation and the ability to read near-print of a certain size gives an indication of 'accommodating' IOL visual function performance, but are influenced by factors such as pupil size, ocular aberrations and an individual's tolerance to blur. [START_REF] Gupta | Optimising measurement of subjective amplitude of accommodation with defocus curves[END_REF] Objective accommodation in 'accommodating' IOL evaluation studies has been assessed by dynamic/streak retinoscopy, lens movement (assessed by ultrasound or partial coherence interferometry) to a pharmacologically induced ciliary muscle contraction or a contralateral physiological accommodative target, autorefraction or aberrometry [START_REF] Wolffsohn | Objective accommodative amplitude and dynamics with the 1CU 'accommodative' intraocular lens[END_REF]. Retinoscopy relies on the subjective responses of skilled examiner and often requires additional lenses in front of the eye to quantify the results. IOL movement from pharmacological ciliary muscle contraction appears to give the maximum possible accommodative response, although pharmacologically-induced accommodation does not relate well to natural physiological eye focus [START_REF] Koeppl | Comparison of pilocarpine-induced and stimulus-driven accommodation in phakic eyes[END_REF]. Although the accommodative response is usually similar in both phakic eyes [START_REF] Thorn | Consensual accommodation[END_REF] allowing contralateral stimulation of accommodation to be effective, this cannot be presumed in the pseudophakic eye, where it will be influenced by factors such as IOL size, IOL position, remaining lens capsule elasticity and any capsular fibrosis.

Autorefractors have the advantage of being non-contact, allowing physiological accommodative stimulation of the eye being assessed and are objective and do not affect the patients view of the target through the use of infra-red light [START_REF] Wolffsohn | Continuous measurement of accommodation in human factor applications[END_REF]. However, many are not open-field, potentially resulting in proximal accommodation (also known as instrumentmyopia) [START_REF] Hennessy | Instrument myopia[END_REF], and none have been validated against subjective refraction on a pseudophakic Wolffsohn et al.

-4 -population. Although open-field autorefractors have been extensively validated on a phakic population [START_REF] Mallen | Clinical evaluation of the Shin-Nippon SRW-5000 autorefractor in adults[END_REF][START_REF] Davies | Clinical evaluation of the Shin-Nippon NVision-K 5001 autorefractor[END_REF][START_REF] Hunt | Evaluation of the measurement of refractive error by the PowerRefractor: a remote, continuous and binocular measurement system of oculomotor function[END_REF][START_REF] Cleary | Diagnostic accuracy and variability of autorefraction by the Tracey Visual Function Analyser and the Shin-Nippon NVision-K 5001 in relation to subjective refraction[END_REF][START_REF] Sheppard | Clinical evaluation of the Grand Seiko Auto Ref/ Keratometer WAM-550[END_REF], IOL materials are generally of higher refractive index than the crystalline lens and, therefore, more disparate from that of the aqueous humour, thus potentially increasing the prevalence of surface reflections. These reflections could hinder the autorefractor image analysis and hence distort the measurement [START_REF] Glasser | Restoration of accommodation: surgical options for correction of presbyopia[END_REF]. Win-Hall and Glasser verified the calibration of the WR-5100K (Grand Seiko Co., Ltd, Hiroshima, Japan) autorefractor (also marketed as the NVision-K autorefractor by Shin-Nippon, Commerce Inc., Tokyo, Japan) and iTrace aberrometer using soft contact lenses in 10 pseudophakes [START_REF] Glasser | Objective accommodative measurements in pseudophakic subjects using and autorefractor and aberrometer[END_REF], but only on patients implanted with a spherical IOL and not against subjective refraction. As such, this study was designed to assess the accuracy of the open-field NVision-K / WR-5100K autorefractor against subjective refraction in patients implanted with spherical, aspheric and 'accommodating' IOLs.

METHODS

Informed consent was obtained from the subjects prior to inclusion in the study following explanation of the nature and possible consequences of the study. The research followed the tenets of the Declaration of Helsinki and was approved by the Solihull Local Research Ethics Committee. The inclusion criteria were patients who had undergone routine cataract surgery to remove a lenticular opacity affecting the visual function of the patient, and had been implanted without complication with an IOL at least 3 months previously (maximum 6 months). One-hundred and forty one eyes were assessed, following implantation with one of four IOLs (Table 1).

Table 1:

Implanted IOLs and demographics of patients assessed. The validity of autorefractors is traditionally assessed by comparing their results to those of subjective refraction, which although more variable than objective measures, [START_REF] Bullimore | The repeatability of automated and clinical refraction[END_REF] provides an endpoint of optimum subjective acceptance. Consequently, all subjects underwent a routine 6 metre, non-cycloplegic, refraction performed by one of three qualified optometrists who was masked from the subjects' habitual prescription and the results of the autorefraction.

IOL

Retinoscopy was performed on all subjects followed by cross-cylinder to determine both the axis (in 2.5˚ increments) and power (in 0.25 D increments) of the cylinder component. Best sphere and binocular balancing was used to refine the spherical component of the prescription (in 0.25 D increments), adopting an endpoint criterion of maximum plus consistent with optimum visual acuity.

The assessed autorefractor was the Shin-Nippon NVision-K 5001 (also branded as the Grand Seiko WR-5100K as described earlier) as part of a family of open-field instruments used extensively in accommodative research. These have been validated previously in children and adults and some versions have been converted to allow dynamic measurement of ocular accommodation [START_REF] Mallen | Clinical evaluation of the Shin-Nippon SRW-5000 autorefractor in adults[END_REF][START_REF] Davies | Clinical evaluation of the Shin-Nippon NVision-K 5001 autorefractor[END_REF][START_REF] Sheppard | Clinical evaluation of the Grand Seiko Auto Ref/ Keratometer WAM-550[END_REF][START_REF] Wolffsohn | Dynamic measurement of accommodation and pupil size using the portable Grand Seiko FR-5000 autorefractor[END_REF][START_REF] Wolffsohn | Continuous recording of accommodation and pupil size using the Shin-Nippon SRW-5000 autorefractor[END_REF]. The absence of an internal fixation target or enclosed viewing reduces the risk of proximal accommodation. Refractive error is calculated in two stages. An infra-red light target is imaged after reflection off the retina, permitting refraction measurements from pupils as small as 2.3 mm. Initially, a lens is moved rapidly on a motorized track to place the target approximately in focus. The image is then analysed digitally to calculate the toroidal refractive prescription over a range of ±22.00 D sphere, ±10.00 D cylinder in steps of 0.12 D for power (with adjustable vertex distance).

Autorefraction was performed while the subjects viewed a high contrast Maltese-cross at optical infinity within a Badal optical system. The instrument was aligned with the visual axis of the eye and five consecutive readings were taken. The measures were repeated 2 months after the first.

RESULTS

The residual refractive error of the sample, as represented by subjective refraction, ranged from -3.31 to +2.88 D, mean spherical equivalent (mean = 0.32 ± 1.08 D). The maximum amount of astigmatism was -6.12 DC.

Validity

The NVision-K / WR-5100K autorefractor prescription was similar (mean difference: +0.09 ± 0.53 D, mean spherical equivalent; p = 0.19) to that found by subjective refraction. There was little bias in the accuracy of the mean autorefractor prescription with respect to the sign or magnitude of the refractive error (Figure 1). Approximately 42% of autorefractor measures were within ±0.25 D and 71% within ±0.50 D of the spherical component of the prescription determined by subjective refraction (Figure 2).

The autorefractor horizontal cylindrical component was similar (mean difference: 0.00 ± 0.39 D, p = 0.96) to that found by subjective refraction and there was no apparent bias with respect to the sign or magnitude (Figure 3). However, the oblique (J 45 ) autorefractor cylindrical vector was slightly more negative (by -0.06 ± 0.25 D, p = 0.06) than the subjective refraction and was biased towards the sign of the cylindrical power (i.e. more negative difference between autorefractor and subjective refraction for more negative oblique cylindrical vectors; Figure 3).

The validity of the autorefractor measurements with each of the IOL designs is presented in Table 2. Implantation of the Softec1 resulted in a significantly more hypermetropic difference between autorefractor reading and the subjective refraction mean spherical equivalent than the Tetraflex IOL (F = 72.77, p = 0.04). However, there was no difference in the accuracy of

DISCUSSION

Objective measurement of ocular refraction is key to understanding accommodation and assessing 'accommodating' IOLs. While the use of a wavefront sensor to achieve this is attractive due to their ability to assess the distortion of light across the whole pupil and ability to assess higher order as well as spherical and astigmatic changes in optical power, no open-field, commercially available devices are widely available. Autorefractors, due to their design to assess refractive error through even small pupils, generally measure from limited locations around a small annulus centred on the visual axis, making them susceptible to failure in detection of localised changes in lens power. [START_REF] Wolffsohn | Investigating the mechanism of action of the Tetraflex KH3500 'accommodative' intraocular lens[END_REF] However, autorefractors are less expensive than aberrometers and commonly available in clinic practice. Furthermore, some are open-field, allowing the rapid assessment of the oculomotor response to observed targets. Autorefractors are, therefore, ideally placed for the objective measurement of ocular accommodation.

This study assessed the accuracy against subjective refraction of an open-field autorefractor already used in IOL studies to provide objective measurement of accommodation [START_REF] Wolffsohn | Objective accommodative amplitude and dynamics with the 1CU 'accommodative' intraocular lens[END_REF][START_REF] Wolffsohn | Investigating the mechanism of action of the Tetraflex KH3500 'accommodative' intraocular lens[END_REF].

Despite concerns that IOLs materials could cause reflections that could complicate the autorefractor and aberrometer analysis [START_REF] Glasser | Restoration of accommodation: surgical options for correction of presbyopia[END_REF], the findings build on a calibration study on spherical IOLs [START_REF] Glasser | Objective accommodative measurements in pseudophakic subjects using and autorefractor and aberrometer[END_REF], showing that the Shin-Nippon NVision-K 5001 / Grand Seiko WR-5100K can provide repeatable results that are similar to subjective refraction in pseudophakes implanted with spherical, aspheric and 'accommodating' IOLs. There is no 'gold standard' against which an autorefractor can be assessed on patients implanted with accommodating IOLs attempting to focus at different distances to compare the results. This study has shown that the autorefractor can accurately measure the optical power of the eye over a range of different refractive powers (mean spherical equivalent -3.8 to +2.9D as assessed by subjective refraction) and hence patients changing their eye focus over this range would be accurately detected. This has been indicated in our previous studies when the autorefractor has been able to detect static optical changes in accommodating IOL power over a 4.0D stimulus response curve and even in dynamically tracing the accommodative response of eyes following a target moving towards and away from them. [START_REF] Wolffsohn | Objective accommodative amplitude and dynamics with the 1CU 'accommodative' intraocular lens[END_REF][START_REF] Wolffsohn | Investigating the mechanism of action of the Tetraflex KH3500 'accommodative' intraocular lens[END_REF] Interestingly, in contrast to the small myopic shift in the spherical and aspheric IOLs, the 'accommodating' IOLs both gave more hypermetropic results than the subjective refraction, perhaps due to flexure of the haptics or optic as has recently been shown. [START_REF] Wolffsohn | Investigating the mechanism of action of the Tetraflex KH3500 'accommodative' intraocular lens[END_REF] It might have been predicted that the aspheric IOL would have resulted in a more myopic bias compared to subjective refraction compared to the other IOLs due to its power profile change across the lens, but within the autorefractors assessment of power around an annulus of 1.5 mm radius from the visual axis, [START_REF] Mallen | Clinical evaluation of the Shin-Nippon SRW-5000 autorefractor in adults[END_REF][START_REF] Davies | Clinical evaluation of the Shin-Nippon NVision-K 5001 autorefractor[END_REF] this was not evident.

This study confirms autorefraction as a simple and valid method of assessing objective 'accommodation' without the limitations of corneal contact, pharmacological stimulation, [START_REF] Koeppl | Comparison of pilocarpine-induced and stimulus-driven accommodation in phakic eyes[END_REF] and presumed contralateral stimulation of accommodation in pseudophakes. Hence future studies of IOLs claiming an 'accommodative' ability can include an assessment of objective changes of focus as well as perceived patient benefits and subjective range of focus, allowing a better understanding of their mechanism of action.

Figure 1 :

 1 Figure 1:The average mean spherical equivalent compared to the difference for the autorefractor versus subjective refraction (black) and the autorefractor repeated on 2 occasions separated by 2 months (grey). Dashed line indicates 95% confidence interval.

Figure 2 :

 2 Figure 2:Histogram of the difference in mean spherical equivalent between the autorefractor and subjective refraction.

Figure 3 :

 3 Figure 3:The average versus the difference between the autorefractor and subjective refraction (black) and the autorefractor repeated on 2 occasions separated by 2 months (grey) for cylindrical J 0 vector (square) and J 45 vector (triangle) component. Dashed line indicates 95% confidence interval.

Acknowledgements:

The authors gratefully acknowledge access to their patients given by Mark Benson and Ian Cunliffe of the Midland Eye Institute, Birmingham, UK

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group

Statistical analysis

If both eyes had been implanted, only the data from the right eye was used. Due to the inherent problems of analysing cylindrical components in their conventional form, [START_REF] Bullimore | The repeatability of automated and clinical refraction[END_REF] sphere, cylinder and axis were converted into a vector representation: [START_REF] Thibos | Power vectors: an application of fourier analysis to the description and statistical analysis of refractive error[END_REF] a spherical lens of power MSE (equal to the mean spherical equivalent = sphere + [cylinder / 2]); a Jackson crosscylinder at axis 0˚ with power J 0 (= -[cylinder / 2] cos[2 x axis]); and a Jackson cross-cylinder at axis 45˚ with power J 45 (= -[cylinder / 2] sin[2 x axis]). The MSE power is typically used to assess changes in refraction with accommodation. [START_REF] Wolffsohn | Objective accommodative amplitude and dynamics with the 1CU 'accommodative' intraocular lens[END_REF] These biases between measures (the mean difference, its standard deviation and 95% confidence intervals) were calculated as suggested by Bland and Altman. [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] The differences between the NVision-K / WR-5100K autorefractor prescription were calculated with paired t-tests and between intraocular lenses with analysis of variance. Intra-session repeatability was obtained by averaging the 95% confidence intervals between the five readings taken for each individual. Inter-session repeatability measurements were calculated from the difference between the original results and the measures taken 2 months later.

-9 -the autorefractor compared to subjective refraction with either of the cylindrical vectors (J 0 : F= 2.01, p = 0.12; J 45 : F = 2.16, p = 0.10). ---------------Insert Figures 123about here---------------

Repeatability

The average intra-session repeatability (95% confidence interval of 5 readings) was 0.54 D for the mean spherical equivalent, 0.48 D for the J 0 vector and 0.29 D for the J 45 component of the prescription. The difference between the average autorefractor measurements for each subject (inter-session repeatability -repeated measurement separated by 2 months) was -0.14 ± 0.87 D (average ± 95% confidence interval) with 85% within 0.50 D for the mean spherical equivalent (Figure 1), -0.01 ± 0.51 D with 96% within 0.50 D for the J 0 vector and -0.03 ± 0.59 D with 96% within 0.50 D for the J 45 component of the prescription. Repeatability was similar between the IOL designs (p > 0.05).

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Competing Interests:

None declared


M.D Nina Hautala
email: nina.hautala@ppshp.fi

Heikki Kauma

Olli Vapalahti

Saara-Mari Mähönen

Olli Vainio

Antti Vaheri

Timo Hautala

Prospective study on ocular findings in acute Puumala hantavirus infection in hospitalized patients

Keywords: hantavirus, intraocular pressure, myopic shift, nephropathia epidemica

Aims: To appraise the ocular manifestations of Puumala hantavirus evoked haemorrhagic fever with renal syndrome (HFRS) nephropathia epidemica (NE) and to clarify the mechanisms of ocular changes in the largest series of patients examined to date.

Methods: 92 eyes of 46 patients with serologically proven NE were examined during the acute phase and after clinical recovery. Ocular symptoms were recorded and visual acuity, refraction, intraocular pressure and ocular dimensions were evaluated.

Results: 88% of the patients experienced decreased intraocular pressure (IOP) (p<0.001), 87% blurred reduced visual acuity, 87% conjunctival chemosis, 82% thickening of the lens (p<0.05), 78% myopic shift (p<0.001), 64% shallowing of the anterior chamber (p<0.05) and 52% shallowing of vitreous length (p<0.05) during the acute phase compared to that measured after clinical recovery. In all, 70% of the patients reported ocular symptoms.

Conclusion

: Ocular symptoms and disturbances are common in acute NE and the symmetry of the ocular changes reflects the systemic nature of the disease. A decrease in IOP and myopic shift mainly due to thickening of the lens are evident in the acute disease. The myopic shift only partially explains the visual disturbances supporting the possible multifactorial origin of the ocular findings in patients with NE.

INTRODUCTION

Haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses is common in Europe and Asia [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Kariwa | Hantavirus infection in East Asia[END_REF]. Puumala hantavirus (PUUV) causes HFRS also called nephropathia epidemica (NE) which is widely found, especially in Northern Europe [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. In Finland, for example, 5% of the population have antibodies against PUUV [START_REF] Brummer-Korvenkontio | Epidemiological study of nephropathia epidemica in Finland 1989-96[END_REF]. Recently, an increasing number of NE cases has been detected in Central and Western Europe, possibly due to climate change [START_REF] Tersago | Hantavirus disease (nephropathia epidemica) in Belgium: effects of tree seed production and climate[END_REF]. The virus is spread by bank voles and there is a cyclic variation in incidence of NE with peaks in every third to fourth year. Human infection is thought to occur by inhalation of the aerosolized excreta of infected rodents. After an incubation period of 2-4 weeks, the typical symptoms of NE start with an acute onset of fever, headache, abdominal pain, nausea, diarrhea, impaired renal function, reduced blurred vision, and occasionally haemorrhagic complications. Although most patients recover from NE spontaneously, a few deaths have been reported [START_REF] Forslund | Complications of nephropathia epidemica: three cases[END_REF][START_REF] Hautala | Hypophyseal hemorrhage and panhypopituitarism during Puumala virus infection: magnetic resonance imaging and detection of viral antigen in the hypophysis[END_REF] and involvement of the central nervous system (CNS) during acute NE has been demonstrated [START_REF] Hautala | Hypophyseal hemorrhage and panhypopituitarism during Puumala virus infection: magnetic resonance imaging and detection of viral antigen in the hypophysis[END_REF][START_REF] Alexeyev | Neurological manifestations of hemorrhagic fever with renal syndrome caused by Puumala virus: review of 811 cases[END_REF][START_REF] Toivanen | Acute disseminated encephalomyelitis following nephropathia epidemica[END_REF].

The ocular symptoms associated with acute fever, renal failure, and thrombocytopenia should lead to a suspicion of NE, at least in endemic regions. Despite the highly characteristic nature of the reduced blurred vision in NE, our understanding of the ocular symptoms remains incomplete.

Reduced Blurred vision (6-40%), transient myopia (8-53%) or myopic shift (19-41%), change in ocular dimensions, acute glaucoma attacks (0-23%), decrease in intraocular pressure (0-66%), anterior uveitis (0-24%), lid edema (0-77%), conjunctival chemosis (3-57%), retinal edema (0-15%), conjunctival and retinal haemorrhages (0-7%), diplopia (0-15%), pupillary defect (single case), and anisocoria (0-7 %) have been reported in patients with NE [START_REF] Techow | Akkomodationskrampf als Krankenheitssymptom bei schlammfieberähniclicher Erkrankung. Eine erstemalige und neuartige Beobachtung[END_REF][START_REF] Lähdevirta | Nephropathia epidemica in Finland: A clinical, histological and epidemiological study[END_REF][START_REF] Saari | Silmämuutokset epideemisessä nefropatiassa[END_REF][START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF][START_REF] Saari | Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome[END_REF][START_REF] Laatikainen | Acute posterior multifocal placoid pigment epitheliopathy in connection with acute nephritis[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Pärssinen | Tonic pupillary reaction after epidemic nephropathy and transient myopia[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF][START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF][START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF][START_REF] Ahlm | Central nervous system and ophthalmic involvement in nephropathia epidemica (European type of haemorrhagic fever with renal syndrome)[END_REF]. However, it is difficult to understand the mechanisms of these ocular symptoms since often the findings are based on case reports [START_REF] Techow | Akkomodationskrampf als Krankenheitssymptom bei schlammfieberähniclicher Erkrankung. Eine erstemalige und neuartige Beobachtung[END_REF][START_REF] Lähdevirta | Nephropathia epidemica in Finland: A clinical, histological and epidemiological study[END_REF][START_REF] Saari | Silmämuutokset epideemisessä nefropatiassa[END_REF][START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF][START_REF] Saari | Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome[END_REF][START_REF] Laatikainen | Acute posterior multifocal placoid pigment epitheliopathy in connection with acute nephritis[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF] or studies involving groups of patients in very different clinical stages of the disease or different degrees of severity [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Pärssinen | Tonic pupillary reaction after epidemic nephropathy and transient myopia[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF][START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF][START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF]. For example, controversy exists in understanding of the possible changes in intraocular pressure [11-13, 15, 16, 19] as well as in occurrence of anterior uveitis during the acute phase of the disease [START_REF] Lähdevirta | Nephropathia epidemica in Finland: A clinical, histological and epidemiological study[END_REF][START_REF] Saari | Silmämuutokset epideemisessä nefropatiassa[END_REF][START_REF] Saari | Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF]. Brief reports on ocular symptoms and findings similar to those seen in NE have been described in HFRS caused by other hantaviruses [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Kariwa | Hantavirus infection in East Asia[END_REF].

We evaluated 46 acute-phase NE patients and re-examined them one month later; our goal was to clarify the spectrum of ocular manifestations and to elucidate the mechanisms of these features in this largest series of NE patients to date. One major advantage of this prospective study is that the patients had similar degrees of severity in their NE and the examinations for ocular involvement were completed within strict time limits since the onset of symptoms.

MATERIALS AND METHODS

Patients

The study patients were recruited from those individuals who were hospitalized for acute NE at the Oulu University Hospital in Northern Finland between September 2005 to February 2008. The patient population and the severity of their clinical symptoms has been described elsewhere [START_REF] Hautala | Pituitary injury may complicate acute PUUV hantavirus infection[END_REF].

During the study period, 154 patients with moderate to severe illness and a positive acute-phase PUUV antibody finding had been hospitalized according to the discharge register. Of those patients, a clinician contacted the newly diagnosed patients though some eligable patients may not have been contacted because of absence of the study doctors, short hospitalization, or other technical reasons.

A comprehensive ophthalmic evaluation was conducted in 50 patients (35 men, 15 females) aged 48 years on average (range 26-77 years) in the acute phase being repeated in 46 patients one month later.

Study Protocol and Methods

The study protocol was explained to each patient and they had the right to refuse or withdraw from the study according to the Helsinki Declaration. The study was approved by the Ethics Committee of the Oulu University Hospital and all participants signed an informed consent form.

The ophthalmic evaluation consisted of the determination of refraction and best corrected visual acuity (BCVA), intraocular pressure (IOP) measurements, slit-lamp examination, dilated funduscopy and evaluation of ocular dimensions by ophthalmic echography (Quantel Medical Compact II). The parameters examined were anterior chamber depth (ACD), thickness of the lens, axial length (AXL) of the eye, and length of vitreous cavity (VL). The ultrasound equipment was calibrated and the reliability of the measurements was confirmed by measuring the same parameters (ACD, AXL) also by IOL Master (Zeiss IOL Master Software Version 3.xx). The patients were examined by an ophthalmologist during the acute phase 5-7 (mean 5.9) days after onset of fever and re-examined after 26-52 (mean 36.8) days after the time of clinical recovery.

Statistical methods and data analysis

The results of the ophthalmic examinations were analysed by SPSS (version 13.0, SPSS Inc., Chicago, Ill., USA) using non-parametric tests: the Wilcoxon signed ranks test was used for normally distributed variables and the Mann-Whitney U test for those variables that were not normally distributed. P-value <0.05 was considered statistically significant.

RESULTS

Symptoms, findings and visual acuity

First, the symptoms were recorded (Table 1). In all, 70% of the patients had complained of a noticeable ocular symptom or frequently, several simultaneous symptoms, during the acute phase.

Common symptoms were light sensitivity light sensitiveness, foreign-body sensation and discomfort, and orbital pain. Furthermore, 87% of the patients reported symptoms suggestive of CNS involvement (headache, nausea/vomiting, or dizziness). In the ophthalmic evaluation, prominent conjunctival chemosis was noted in 80 eyes. Only 1 patient suffered bilateral subconjunctival bleeding. No cases of anterior or posterior uveitis, retinal edema or retinal haemorrhages were recorded.

VA was blurred reduced in 80 eyes of 40 patients (87%) during acute NE (Table 1). The mean BCVA was diminished (20/25, range 20/500-20/10) compared to the control assessment (20/16, range 20/30-20/10 (p<0.001)). VA remained unchanged in 9 eyes (10%) and was better in 3 eyes (3%) in the acute phase compared to the control examination.

Refraction

A strong tendency was noted towards myopia in the acute phase compared to control examination (Table 2). If all patients were considered as a single group, the mean change in refraction power

Intraocular pressure

In all patients, the IOP tended to be significantly lower in the acute phase compared to control phase (Table 2). The differences in IOP between RE and LE were parallel in each individual patient. The IOP had declined (mean 4.5 mmHg, range 1-11 mmHg) in 80 eyes of 40 patients (87%) in the acute phase compared to that measured after clinical recovery (p<0.001). In 9 eyes (10%) the IOP remained unchanged during the acute infection and in 3 eyes (3%) a slight increase in IOP (mean 3 mmHg, range 1-5 mmHg) was noted during the acute phase. However, neither of these two patients had IOP over 22 mmHg nor suffered any symptoms of acute angle-closure attack in the acute phase.

IOP was measured at the same time of day in each patient to avoid the diurnal variation.

Anterior chamber depth

Compared to the control phase, mean ACD was shallower in 59 eyes (64%), deeper in 25 eyes (27%) and unchanged in 8 eyes (9%) in the acute phase. The individual difference in ACD was parallel between RE and LE (Table 2).

Lens

Compared to the control examination, in the acute phase deepening of the crystalline lens was noted in 75 eyes (82%), the lens was thinner in 14 of 92 eyes (15%) and no change was noted in 3 eyes. In the acute phase, the average thickness of the lens was significantly increased (Table 2).

Vitreous length and axial length

The length of the vitreous cavity was shallower in the acute phase compared to control phase (Table 2). There was no statistical difference in AL between the phases.

DISCUSSION

Previous reports have examined the ophthalmic aspects of NE mainly in patients with mild or moderate systemic disease [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF][START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF][START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF]. However, controversy exists on some findings, such as changes in IOP, possibly due to variation in time-points at which the ocular findings were recorded during the course of the illness in the earlier studies. In our study, which is the largest series to date concerning ocular involvement in NE, the patients were examined on an average of six days after the onset of symptoms i.e. during the acute febrile and oliguric phase of the illness. They were reexamined soon after their recovery which should rule out the possibility of other potential developing ocular conditions complicating the findings. There are extensive variations in the severity of systemic NE, and this may also influence the ocular manifestations; all our patients experienced moderate or severe disease, which may in part explain the homogenous and consistent results. Interestingly, all our patients displayed ophthalmic findings and 70% of them suffered marked ocular symptoms. It is obvious that ocular involvement is indeed a common feature of acute NE and in actual fact the findings may be more common than formerly believed [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF].

Blurred Reduced vision, which is highly characteristic of NE, is commonly thought to be caused by a myopic shift or transient myopia [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF], although the precise mechanism of visual disturbance has remained unknown. Previous studies have reported the incidence of transient myopia or myopic shift of 8-53% in hospitalized patients [START_REF] Saari | Silmämuutokset epideemisessä nefropatiassa[END_REF][START_REF] Saari | Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF]. Compared to previous studies, the greater incidence of myopic refraction change in the current study was 74%, which may be explained by the severity of the systemic disease in our patients. In addition, our patients were examined during the febrile phase of the illness and the majority of the patients were suffering ocular symptoms at that time point. The symptoms and ocular findings of NE are known to be transient and examination of refraction at much earlier or later time points could well lead to the different results. Interestingly, the change of refraction towards a myopic direction does not necessarily lead to decreased visual acuity in patients with acute phase of NE. Indeed, the myopic shift may even improve the visual acuity of hyperopic patients when not wearing eyeglasses.

An earlier study from Finland has suggested that myopia is caused mainly by shallowing of the anterior chamber [START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF]. In our patients, the shallower anterior chamber was shallower in 64% of the patients in the acute NE and this may partly explain the myopic change in some but not all patients displayed a myopic refraction change. Interestingly, the anterior chamber was deeper in 27% of the patients in the acute phase and some of these patients had a simultaneous myopic change. This finding does not support the previous hypothesis that the depth of the anterior chamber plays a major role in the mechanism of myopia [START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF]. Another commonly discussed feature claimed to account for the myopic shift is thickening of the lens [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF]. In our study, thickening of the lens was noted in 82% of all NE cases encompassing the patients with a myopic refraction change. The increased thickness of the lens evokes a myopic change by steepening the anterior and posterior curvatures of the lens. In addition, the refractive index of the lens may change due to changes in osmolarity of both the lens and the aqueous humor during acute NE. Simultaneously with the thickening of the lens, the length of the vitreous cavity tended to be shallower in acute phase than in the control phase despite the possible forward movement of the lens in acute NE. In contrast to a previous work [START_REF] Kontkanen | Myopic shift and its mechanism in nephropathia epidemica or Puumala virus infection[END_REF], no significant change in the axial length of the eyes in the acute phase was noted. These results suggest that thickening of the lens seems to be the key factor in the mechanism contributing to the myopic change in acute NE.

Contradictory results of IOP level in patients with NE have been published [START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF][START_REF] Neetens | Pathogenie de l'elevation de la pression intra-oculaire dans la nephropathie epidemique[END_REF] and there are some reports of acute glaucoma attacks associated with NE [START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Neetens | Pathogenie de l'elevation de la pression intra-oculaire dans la nephropathie epidemique[END_REF]. The increased IOP has been speculated to be due to edema and haemorrhage in the ciliary body, its anterolateral rotation, relaxation of the zonules and the anterior movement of the lens [START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF]. There are several reports of decreased IOP during acute NE [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF], although a case of an acute glaucoma attack was documented in one of these series of patients [START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF]. In a previous work, IOP declined in 66 % and increased or remained unchanged in 34 % of the patients, whereas the mean differences in IOP between the phases were less than 2 mmHg 1.8 mmHg in the right and 1.9 mmHg in the left eyes [START_REF] Kontkanen | Intraocular pressure changes in nephropathia epidemica: A prospective study of 37 patients with acute systemic Puumala virus infection[END_REF]. In agreement with these works, our results indicate an even more significant (4.0 mmHg and 3.6 mmHg in the right and left eyes, respectively) decrease in IOP between the phases. In contrast to some previous studies [START_REF] Saari | Acute glaucoma in haemorrhagic fever with renal syndrome (nephropathia epidemica)[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Neetens | Pathogenie de l'elevation de la pression intra-oculaire dans la nephropathie epidemique[END_REF], there were no cases of acute angle-closure attack in our patients. We thus conclude that decreased IOP is a general characteristic of acute NE. However, we can not rule out the possibility that the observed change may not be specific for NE; potential IOP alterations during other febrile illnesses are not well understood.

It is known that the acute phase of NE causes increased tissue permeability [START_REF] Kanerva | Pathogenesis of Puumala and other hantavirus infections[END_REF]. Leakage of erythrocytes has been described as interstitial hemorrhages in many organs including conjunctiva and retina [START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF] which could explain the finding of subconjunctival bleeding and frequent conjunctival chemosis in our patients. In some previous studies, 7-24% of the several patients have been diagnosed with anterior uveitis during the acute NE [START_REF] Saari | Silmämuutokset epideemisessä nefropatiassa[END_REF][START_REF] Saari | Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome[END_REF][START_REF] Saari | Ophthalmological findings in nephropathia epidemica in Lapland[END_REF][START_REF] Pärssinen | Ophthalmic manifestations of epidemic nephropathy[END_REF][START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF]. On the contrary, no cases of uveitis connected to acute NE were recorded in our study. Interestingly, a prior study has reported that uveitis had subsided without the need for a medical therapy [START_REF] Kontkanen | Ocular characteristics in nephropathia epidemica or Puumala virus infection[END_REF], suggesting that the increase in tissue permeability in the capillary endothelial cells of the ciliary body may have triggered a condition resembling uveitis rather than actual inflammation of the uvea. We speculate that edema and hemorrhages in the ciliary body may cause decreased aqueous formation and diminished filtration due to damage to the capillary endothelial cells which would subsequently lead to the decreased IOP. It is possible that the decreased filtration of the ciliary body and the simultaneous disturbed kidney function may share similar biological mechanisms.

In our study, visual acuity was diminished in 87% of the patients with acute NE. This cannot be completely explained by myopization, which was documented in 74% of the cases. Thus, 18% of the patients had no change in the refraction power but most patients still suffered from reduced blurred vision during the acute phase. In some of these patients, normal vision was not achievable even with best spectacle correction and there were no signs of other features, i.e. retinal edema, haemorrhages or uveitis, which would possibly account for the impaired visual acuity. This interesting feature leads us to conclude that the visual disturbances encountered in acute NE can be caused by multiple mechanisms. Extraocular origins, such as CNS involvement, cannot be ruled out in patients with severe NE as a potential explanation of the ocular visual changes. In order to clarify these mechanisms, for example, the role of CNS involvement during acute NE should be investigated.

COMPETING INTEREST

None declared.

LICENCE FOR PUBLICATION

I, Nina Hautala, The Corresponding Author of this article has the right to grant on behalf of all authors and does grant on behalf of all authors, a licence to the BMJ Publishing Group Ltd and its licensees, to permit this Contribution (if accepted) to be published in British Journal of Ophthalmology (BJO) and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at: (http://bjo.bmj.com/site/about/licence.pdf).

Rev Med Virol. 1998;8:67-86.

FIGURES AND TABLES

Table 1. The incidence of ocular symptoms and findings in patients with acute NE.

  was 0.5 D towards myopic direction in RE (range from -4.5 D to +0.75 D, p<0.001) and 0.5 D in LE (range from -4.5 D to +0.75 D, p<0.001). Myopic refraction change (mean -0.7 D, range 0.25-4.5 D, p<0.001) was noted in a total of 68 eyes of 34 patients (74%). Refraction remained unchanged in 17 eyes (18%) and in 7 eyes (8%) refraction changed in the hyperopic direction (mean +0.4 D, range 0.25-0.75 D). The changes in refraction power were analogous between RE and LE in an individual patient.

Table 2 .

 2 Ocular findings in acute NE compared to control phase. The results are expressed as mean (SD). IOP = intraocular pressure (mmHg), ACD = anterior chamber depth (mm), VL = vitreous length (mm), AL = axial length (mm). Refraction power is expressed as dioptres (D). RE represents right eye and LE left eye. A p-value <0.05 is considered statistically significant, * p<0.05 and ** p<0.001.

Table 1 . Symptoms and findings Number of patients (%)

 1

	Blurred visual acuity	40 (87%)
	Conjunctival chemosis	40 (87%)
	Headache, nausea/vomiting, or dizziness	40 (87%)
	Light sensitiveness	22 (48%)
	Foreign-body sensation	13 (28%)
	Orbital pain	8 (17%)
	Subconjunctival bleeding	1 (2%)

Table 2 .

 2

	Acute phase
	RE	10.9 (2.9)	-1.0 (1.9)	2.9 (0.6)	4.6 (0.5)	15.5 (1.0)	23.1 (0.8)
	LE	11.1 (3.3)	-0.7 (1.7)	2.8 (0.5)	4.5 (0.5)	15.6 (0.8)	23.0 (0.9)
	Control phase
	RE	14.9 (2.8)	-0.5 (1.8)	3.1 (0.5)	4.3 (0.4)	15.8 (1.0)	23.1 (1.1)
	LE	14.7 (2.7)	-0.2 (1.5)	3.2 (0.4)	4.2 (0.4)	15.8 (0.9)	23.1 (1.0)
	Difference between the phases
	RE	4.0 **	0.5 **	0.2 *	0.3 **	0.3 **	0.1
	LE	3.6 **	0.5 **	0.4 **	0.3 **	0.2 *	0.1

IOP (mmHg) Refraction (D) ACD (mm) Lens (mm) VL (mm) AL (mm)

* p<0.05, ** p<0.001