Alfredo Ruggeri #
email: alfredo.ruggeri@unipd.it

Fabio Scarpa #

Massimo De Luca #

Christian Meltendorf

Jan Schroeter

Prof Alfredo Ruggeri

A system for the automatic estimation of morphometric parameters of corneal endothelium in alizarine red stained images

Keywords: morphometry, cornea endothelium, alizarine red, image analysis. Word count (excluding title page, abstract, tables and figures

Background/aims. A computer program for the automatic estimation of morphometric parameters (cell density, pleomorphism, polymegethism) in alizarine red stained images is presented and evaluated.

Methods. Images of corneal endothelium from 30 porcine eyes stained with alizarine red were acquired with an optical microscope and saved as grey-level digital images. Each image was at first pre-processed for luminosity correction and contrast enhancement. An artificial neural network was used to classify all pixels as cell contour or cell body pixels. The segmented cell contours were then used to obtain estimates of morphometric parameters. The central area was assessed and the mean area per cornea was 0.54±0.07 mm 2 . The whole system was implemented as a computer program using the Matlab ® language. Estimated parameters were compared with the corresponding values derived from manual contour detection on the same images used for the automatic estimation.

Results. On the 30 images of our data set, mean differences of automatic parameters vs. manual ones were -12±52 cells/mm 2 (range -103 to +145) for density; 0.5±2.6 per cent (range -5.6 to +5.6) for pleomorphism; -0.7±1.9 per cent (range -4.1 to +2.8) for polymegethism.

Conclusion. The evaluation of the automatic system on 30 images from porcine eyes confirmed its capability of reliably estimating morphometric parameters with respect to parameter values derived by manual analysis.

INTRODUCTION

The evaluation of the main morphometric parameters of corneal endothelium provides information to assess the cornea health state. Cell density (ECD), polymegethism, i.e., differences in cell size often expressed as coefficient of variation (or fractional standard deviation) of cell areas, and pleomorphism (or hexagonality coefficient), defined as the fraction of hexagonal cells over the total number of cells, allow the quantitative characterization of the endothelial cell conditions. All these parameters require the reliable identification of cell contours in endothelial images. Computer programs have been proposed to accomplish this task since almost 30 years ago, see e.g. [START_REF] Lester | Automated morphometric analysis of corneal endothelial cells[END_REF][START_REF] Koyama | Image analysis of morphologic features of the corneal endothelium including hexagonality[END_REF][START_REF] Oblak | A semi-automated assessment of cell size and shape in monolayers, with optimal adjustment for the cell-cell border width -application to human corneal endothelium[END_REF][START_REF] Giasson | Morphometry of cells and guttae in subjects with normal or guttate endothelium with a contour detection algorithm[END_REF][START_REF] Siertsema | Automated video image morphometry of the corneal endothelium[END_REF], even if at the best of our knowledge all such programs only provide a semi-automatic analysis.

In order to increase cell contour visibility, and thus to allow a more reliable estimation of the morphometric parameters above, dyes such as alizarine red are used in many in-vitro studies. In one such study, the quality control in an eye bank setting was carried out to validate the entire process from cornea procurement up to transplantation [START_REF] Schroeter | Influence of temporary hypothermia on corneal endothelial cell density during organ culture preservation[END_REF]. An uncontrolled change of temperature, which may e.g. occur during transportation, can actually exert a potentially harmful non-physiological influence on the corneal endothelial cells [START_REF] Madden | Methods of refrigerated transport of corneas for transplantation[END_REF]. In order to study the effects of significant changes of the storage temperature in organ cultured corneas, an investigation has been performed, where alizarine red stained images were acquired and analyzed by corneal experts, who manually outlined, i.e. segmented, the cell contours to derive an estimation of the morphometric parameters [START_REF] Schroeter | Influence of temporary hypothermia on corneal endothelial cell density during organ culture preservation[END_REF].

Although in stained images cell contours are more visible and better outlined, their manual tracing, albeit performed in a computer-assisted manner, still remains a very long and tedious task. This often leads the experimenter to reduce the number of outlined cells to just a few tens, greatly affecting the accuracy of estimated parameters and thus the reliability of the study outcomes.

To overcome this situation, we have developed and evaluated a totally automatic computerized system that allows the reliable estimation of endothelium morphometric parameters in alizarine red stained images.

MATERIAL AND METHODS

Image acquisition

The corneas were obtained from commercially slaughtered pigs. The corneo-scleral discs were prepared under sterile conditions after disinfections in the laminar airflow unit: 8 mm central corneal disc were prepared using a corneal punch device (Katena Products, Denville, NJ, USA) and then transferred with the endothelium turned upward to a 50 ml polystyrene container (Gosselin Plastiques, Hazebrouck Cedex, France) with 20 ml dextran-containing culture medium (100 ml MEM with Earle´s salts (10x); 20 ml fetal bovine serum; 10 ml penicillin/streptomycin 10.000 U/10.000 µg/ml; 10 ml amphotericin B 250 µg/ml; 10 ml L-glutamine (200 mM); 12.5 ml HEPES buffer (1 M); 29.3 ml sodium bicarbonate (NaHCO3) (7.5 %); 60 g Dextran 500, 748.2 ml aqua ad injectionem). The container was then closed air-tight and stored at 32°C for 24 hours.

Endothelial cell borders were visualized by staining with 0.2% alizarin red (Waldeck/Chroma, Münster, Germany) for 4 minutes. The pH of alizarin red was adjusted to 4.2 by using 0.1% ammonia solution [START_REF] Taylor | Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction[END_REF]. The dye was then rinsed with 0.9% sodium chloride solution. Previous removal of the scleral ring enabled us to place the corneal discs on the microscope slides with the endothelium pointing downwards, and thus to display the endothelium on one level. Endothelium images were obtained from 30 of such pig corneas using a calibrated inverse phase contrast microscope (CK 40, Olympus, Hamburg, Germany) at 200x magnification and an analog camera (SSC-DC50AP, Sony, Japan), and digitized as 768 x 576 pixels 8 bits gray-level images using the NAVIS software (Nidek Technologies, Padua, Italy). The calibration of the system was done using a standardized microscale (Neubauer chamber). The mean area assessed per cornea was 0.54±0.07 mm 2 (range 0.31 to 0.64 mm 2 ) and was located in the central zone of the cornea, to avoid bias in the cell morphometric evaluation [START_REF] Amann | Increased endothelial cell density in the paracentral and peripheral regions of the human cornea[END_REF]. In Fig. 1, top panel, a representative image (nr. 7) from our dataset is shown.

All 30 images of this dataset are publicly available for download [10].

Automatic estimation of morphometric parameters

A specific algorithm to identify cell contours was developed and implemented as a computer program. The flow diagram of Fig. 2 illustrates the main steps of the image analysis algorithm.

Image pre-processing. In order to exclude the peripheral region of the images, where contrast is lower and often prevents a reliable detection of cell contours, and to keep the number of analyzed cells reasonably low, a region of interest (ROI) was manually chosen in each image, located approximately in the center of the image, and used for both automatic and manual analysis (see below). In each image, this ROI includes on average 305 cells (range 223 to 442 cells), in order to allow a reliable estimate of the morphometric parameters, and covers an average area of 0.101 mm 2 .

The images from corneal microscopes usually present a non-uniform illumination and some pixel noise. In order to cope with these problems, a parabolic correction was at first used to improve luminosity equalization and contrast enhancement. The compensation of the illumination drift was obtained by fitting it with a parabolic function, which is then subtracted from the image, both horizontally and vertically. A band-pass filtering (with cutoff frequencies of 0.02f c and 0.2f c , where f c is the spatial sampling frequency of the image) was then applied to enhance the space frequency components specifically related to cell contour detection and to reduce the ones related to noise. Contrast, especially the one between dark cell boundaries and light cell bodies, is generally very low in this type of images. Local contrast was further enhanced by applying to each image pixel a sigmoid point transformation, centered on the image mean gray-level value.

Cell contour segmentation. A multi-layer feed-forward artificial neural network (ANN), with local shift invariant interconnections [START_REF] Long | Effective automatic recognition of cultured cells in bright field images using Fisher's linear discriminant preprocessing[END_REF], was used to obtain a binary classification of all image pixels as either cell contour or cell body pixels. Each single pixel of the image was considered and its 121 adjacent pixels (11x11 pixels square neighborhood) were used to extract 7 features, used to classify the pixel as belonging to one of 8 possible classes: cell body; vertical border; horizontal border; oblique border; border with angle towards left; border with angle towards right; border with angle up; border with angle down. Finally, each pixel was classified into cell body (if assigned to the first class) or cell boundaries (if assigned to any of the other 7 classes). The adopted network is composed of 1 input, 2 hidden, and 1 output layers, with 7, 7, and 1 nodes respectively.

The ANN was trained on a set of 5 samples, randomly selected from different images of the dataset; each sample is made of a pair of 120x120 pixels sub-images: the original sub-image, cropped from the acquired image, and the same sub-image with the cell contour manually outlined by a cornea imaging expert.

Cell contour post-processing. The resulting ANN, trained as described above, was applied to the images to obtain a first segmentation of cell contours. This segmented image was then skeletonised [START_REF] Gonzalez | Digital Image Processing[END_REF] to accurately extract the cell contours as 1-pixel thick lines. Unfortunately, grey-level information alone is often not sufficient to correctly detect cell contours. To cope with the errors still present in the segmented image (missing contours or false contours), a postprocessing procedure was developed based on an automatic, multi-step approach, which includes missing contour recovery and tentative merging and splitting of cell bodies. For every identified cell, a set of features was evaluated, such as mean radius, standard deviation of radius, standard deviation of relative radius (radius / mean radius), aspect ratio (ratio of the two main diagonals).

Trying to improve an overall 'cell score', based on the values of these features, attempts were made to fuse small adjacent cells or to split large cells. This was done according to the general paradigm of trying to obtain cells whose shape is close to that of regular polygons: any operation (splitting or fusion) that improves this regularity is accepted. No manual correction of any type was applied to the obtained cell contours.

Computation of morphometric parameters. From the contour images eventually obtained, area and number of sides of all detected cells were straightforwardly derived. From these values, the estimation of ECD, pleomorphism, and polymegethism was finally carried out. ECD was computed as the sum of individual cell areas divided by the total number of cells; pleomorphism was computed by counting for each cell the number of neighboring cells (cells on the border of the considered ROI were excluded from this computation) and taking the percentage of cells with hexagonal shape; polymegethism was computed as the fractional standard deviation of all cell areas.

The whole system was developed as a collection of computer program modules, implemented using the Matlab ® computer language (Matlab v. R2008, http://www.mathworks.com/).

Manual estimation of morphometric parameters

In order to assess the accuracy of the morphometric parameters estimated by the computerized procedure described above, ground truth reference values were obtained on the same ROIs with a manual estimation of the parameters performed by a cornea imaging expert.

Using a public-domain image manipulation program (GIMP v. 2.6, http://www.gimp.org/) on screen-displayed enlarged version of the images, all visible cell contours were drawn, so as to outline the polygonal shape of each cell. The same procedure used in the automatically segmented images (see above) was then used to estimate the morphometric parameters also in the manually segmented ones.

All the manually segmented images of this dataset are publicly available for download [10].

RESULTS

Table 1 reports the statistics for the three morphometric parameters using the manual or the automatic method and the differences between the two methods. As regards ECD, these differences have an average value of 12 cells/mm 2 , with a range from -103 cells/mm 2 to +145 cells/mm 2 , respectively. For pleomorphism these values are 0.5 per cent, -5.6 per cent and +5.6 per cent, respectively; for polymegethism they are -0.7 per cent, -4.1 per cent and +2.8 per cent, respectively. The same statistics for the percent differences (with respect to manual values, assumed as reference values) are: 0%, -1% and +3% for ECD; 1%, -11%, and +13% for pleomorphism; -3%, -17%, and +13% for polymegethism, respectively. Figs. 2 shows the scatter-plots of manually vs. automatically estimated parameters. From this graphical representation of the results, the correlation between the automatic and manual method can be appreciated and quantitatively described by the Pearson's correlation coefficient, with values of 0.99 for ECD and 0.80 for both pleomorphism and polymegethism. To display the extent of agreement between the two methods, Bland-Altman plots [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF][START_REF] Bland | Measuring agreement in method comparison studies[END_REF] of differences vs. averages for each pair of manual and automatic parameters are shown in Fig. 3, where lines indicate the average difference and the 95% limits of agreement: 11.9, -90.4 and 114.3 cells/mm 2 for ECD; 0.48, -4.54 and +5.50 per cent for pleomorphism; -0.69, -4.33, +2.95 per cent for polymegethism, respectively.

One representative example of the contours obtained by the proposed algorithm is shown in Fig. 1, bottom panel. The average run time of the current Matlab ® prototype on a single image is 3 minutes.

DISCUSSION AND CONCLUSION

We have presented here a system for the estimation of cornea endothelium morphometric parameters that is completely automated and requires no manual editing by the user. The estimates of the parameters provided by the proposed algorithm are in very good agreement with the reference ones, obtained with careful manual analysis.

The estimation of ECD can nowadays be performed with reasonably good accuracy with some computerized analysis systems, see e.g. [START_REF] Ruggeri | A new system for the automatic estimation of endothelial cell density in donor corneas[END_REF][START_REF] Gain | Automated tri-image analyser of stored corneal endothelium[END_REF], as the possible presence of a few errors in cells/contours detection is of limited impact on the final ECD estimation. On the contrary, as already noted by other authors, e.g. [START_REF] Doughty | Further analysis of assessments of the coefficient of variation of corneal endothelial cell areas from specular microscopic images[END_REF], the quantitative estimation of pleomorphism and polymegethism is much more affected than ECD by the wrong contour detection even in very few cells, rendering the reliable estimation of these parameters quite difficult. A possible solution is the manual correction of cell borders, but this might require a significant amount of work, e.g., on about 50 to 75 per cent of cell borders [START_REF] Doughty | Further analysis of assessments of the coefficient of variation of corneal endothelial cell areas from specular microscopic images[END_REF]. For the system we propose, the 95% limits of agreement for pleomorphism and polymegethism estimation were always below 5 per cent (in actual calculated value), making the results automatically provided by the system reliable enough to reach clinically significant conclusions. It must be noted, however, that at variance with other proposed systems, contour recognition in this in-vitro study was easier than in in-vivo clinical situations, where a dye cannot be used to increase cell visibility. For this reason, the present version of our system can be used only in experimental settings and not in cornea banking or clinical applications on patients.

In large experimental studies employing alizarine red dye, such as the one addressed here, hundreds of images may need to be analyzed. In this case, for the practical reasons mentioned above, the experimenter usually restricts him/herself to estimate only ECD. The system we propose may thus be extremely valuable, as it can provide the experimenter with reliable estimation of the other two important morphometric parameters. Moreover, with respect to routine manual or semi-automatic analysis, where usually just a few tens of contiguous cells are considered, the proposed algorithm can process images containing hundreds of contiguous cells, to provide higher accuracy for the estimated morphometric parameters. This latter can be further increased by selecting in the central part of the cornea many images, positioned at slightly different locations, each one with hundreds of cells.

The present version of the proposed system, still a research prototype, would require a sizable amount of time to perform such a multi-image analysis. Implementation of the whole system in the more efficient computer language C++ would reduce the execution time to the order of 10 seconds per image, allowing multi-image analysis to be performed within one minute. In addition, in this latter version a user-friendly editing interface might be included, to allow the user to quickly manually correct the possible residual errors in contour detection (on average, in 1 per cent of cells only) and reach a 100% accuracy in parameter estimation (with respect to manual analysis).

Albeit the in-vitro studies addressed here are very important to evaluate and compare different experimental situations, they are however very specific and performed less often than clinical applications. In order to provide an automatic evaluation of morphometric parameters also in clinical images, we are developing a version of our system able to analyze endothelium images acquired in patients, e.g. with specular or confocal microscopes.

Competing Interest: Nonedeclared.

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/licenceforms).

Table and figure legends

Table 1. Summary results of automatic parameter estimation. For each cornea, manual and automatic values are reported for all three parameters, as well as their difference ('Diff'), with percent values ('%'), and absolute difference ('Abs'), also with percent values ('%'). For all quantities, average, standard deviation, minimum and maximum values are reported for the 30 cornea images. Cell densities values are in cells/mm 2 , pleomorphism and polymegethism are per cent values. Fig. 4. Scatter-plots of difference vs. average for each pair of manual and automatic ECD (left), pleomorphism (center) and polymegethism (right). The dotted line shows the average difference, the dashed lines show the 95% limits of agreement [START_REF] Gonzalez | Digital Image Processing[END_REF][START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF].

Fig. 1 .

 1 Fig. 1. Original (top panel) and processed (bottom panel) image of alizarine red stained corneal

Fig. 2 .

 2 Fig.2.Flowdiagramofthemainstepsoftheimageanalysisalgorithm.

Fig. 3 .

 3 Fig. 3. Scatter-plot of automatic vs. manual estimates of ECD (left), pleomorphism (center) and

44 46 48 50 52 54 56 58 60 Automatic pleomorphism (%) Manual pleomorphism (%)

	Image	Manual ECD	Automatic ECD	Diff % Abs %	Manual Pleomorphism	Automatic Pleomorphism	Diff % Abs %	Manual Polymegethism	Automatic Polymegethism	Diff % Abs %
	1	4661	4658	-3 0%	3 0%	44.5			45.5		1.0	2% 1.0 2%	25.9	24.0	-1.9 -7% 1.9 7%
	2	4524	4643	119 3% 119 3%	49.8			52.8		3.0	6% 3.0 6%	27.0	25.0	-2.0 -7% 2.0 7%
	3	4676	4701	25 1% 25 1%	46.8			46.6		-0.2	0% 0.2 0%	24.7	27.1	2.4 10% 2.4 10%
	4	3892	3895	3 0%	3 0%	52.3			49.6		-2.7 -5% 2.7 5%	18.9	18.0	-0.9 -5% 0.9 5%
	5	4405	4383	-22 0% 22 0%	46.2			47.1		0.9	2% 0.9 2%	19.5	19.5	0.0	0% 0.0 0%
	6	4131	4062	-69 -2% 69 2%	49.2			47.4		-1.8 -4% 1.8 4%	19.0	16.6	-2.4 -13% 2.4 13%
	7	4230	4188	-42 -1% 42 1%	48.8			51.8		3.0	6% 3.0 6%	17.7	17.1	-0.6 -3% 0.6 3%
	8	4419	4397	-22 0% 22 0%	53.6			52.3		-1.3 -2% 1.3 2%	19.0	19.1	0.1	1% 0.1 1%
	9	4895	4860	-35 -1% 35 1%	48.1			51.6		3.5	7% 3.5 7%	20.3	20.3	0.0	0% 0.0 0%
	10	4587	4592	5 0%	5 0%	49.1			50.9		1.8	4% 1.8 4%	21.6	21.3	-0.3 -1% 0.3 1%
	11	4171	4316	145 3% 145 3%	47.6			45.6		-2.0 -4% 2.0 4%	23.5	24.8	1.3	6% 1.3 6%
	12	3971	4008	37 1% 37 1%	54.1			58.3		4.2	8% 4.2 8%	22.2	25.0	2.8 13% 2.8 13%
	13	3874	3843	-31 -1% 31 1%	43.7			49.3		5.6 13% 5.6 13%	24.5	26.4	1.9	8% 1.9 8%
	14	3759	3710	-49 -1% 49 1%	41.2			41.2		0.0	0% 0.0 0%	23.6	23.1	-0.5 -2% 0.5 2%
	15	3765	3817	52 1% 52 1%	45.5			46.2		0.7	2% 0.7 2%	24.9	23.6	-1.3 -5% 1.3 5%
	16	3703	3724	21 1% 21 1%	51.5			51.8		0.3	1% 0.3 1%	24.8	21.9	-2.9 -12% 2.9 12%
	17	3750	3773	23 1% 23 1%	49.5			46.6		-2.9 -6% 2.9 6%	24.4	24.5	0.1	0% 0.1 0%
	18	3910	3807	-103 -3% 103 3%	51.2			52.4		1.2	2% 1.2 2%	26.9	25.1	-1.8 -7% 1.8 7%
	19	3793	3848	55 1% 55 1%	52.0			50.2		-1.8 -3% 1.8 3%	21.4	24.1	2.7 13% 2.7 13%
	20	4273	4302	29 1% 29 1%	49.4			43.8		-5.6 -11% 5.6 11%	25.5	26.3	0.8	3% 0.8 3%
	21	4310	4379	69 2% 69 2%	48.6			52.6		4.0	8% 4.0 8%	24.8	23.2	-1.6 -6% 1.6 6%
	22	4426	4503	77 2% 77 2% 40 42	47.3 44	46	48	45.3 50	52	-2.0 -4% 2.0 4% 54 56 58 60	25.0	24.8	-0.2 -1% 0.2 1%
	23 24 25 26 27 28 29 30 average sd min	3828 4057 4209 3960 4283 4065 3857 4097 4149 320 3703	3804 4082 4226 3938 4331 4092 3869 4088 4161 330 3710	-24 -1% 24 1% 25 1% 25 1% 17 0% 17 0% -22 -1% 22 1% 48 1% 48 1% 27 1% 27 1% 12 0% 12 0% -9 0% 9 0% 12 0% 41 1% 52 1% 34 1% -103 -3% 3 0% 40 42 Identity line 48.0 50.0 2.0 4% 2.0 4% 44.2 43.8 -0.4 -1% 0.4 1% 48.5 49.3 0.8 2% 0.8 2% 42.5 41.5 -1.0 -2% 1.0 2% 40.2 38.0 -2.2 -5% 2.2 5% 46.9 50.4 3.5 7% 3.5 7% 48.1 47.9 -0.2 0% 0.2 0% 49.5 52.5 3.0 6% 3.0 6% 47.9 48.4 0.5 1% 2.1 4% 3.4 4.3 2.6 5% 1.5 3% 40.2 38.0 -5.6 -11% 0.0 0%	22.9 23.3 27.6 28.4 24.9 22.2 26.3 23.6 23.5 2.8 17.7	18.9 21.5 28.5 25.4 20.8 21.9 23.2 22.6 22.8 3.0 16.6	-4.0 -17% 4.0 17% -1.8 -8% 1.8 8% 0.9 3% 0.9 3% -3.0 -11% 3.0 11% -4.1 -16% 4.1 16% -0.3 -1% 0.3 1% -3.1 -12% 3.1 12% -1.0 -4% 1.0 4% -0.7 -3% 1.6 7% 1.9 8% 1.2 5% -4.1 -17% 0.0 0%
	max	4895	4860	145 3% 145 3%	54.1			58.3		5.6 13% 5.6 13%	28.4	28.5	2.8 13% 4.1 17%
					R = 0.7983	y = 1.00x + 1.44	! Regression
					16	18	20		22		24	26	28
				16
					R = 0.7984	y = 0.86x + 1.89	! Regression

Automatic -manual polymegethism (%)Average polymegethism (%)


Russell Hc

Bradnam Ms

Ophthalmic, clinical and visual electrophysiological findings in children born to mothers prescribed substitute methadone in pregnancy

Keywords: electrophysiology, drug misuse, methadone, nystagmus Word count: 2160

Background and aims: There are growing concerns regarding visual outcome of infants exposed to opiates (including substitute methadone) and/or benzodiazepines in utero. We describe the combined ophthalmology and visual electrophysiology findings in 20 infants and children who had been exposed to substitute methadone and other drugs of misuse in utero.

Methods: This was a descriptive case series of 20 patients, all of whom had been referred to a paediatric visual electrophysiology service because of concerns regarding visual function, and all of whom had been exposed to methadone in utero. All children underwent a full ophthalmic and orthoptic examination as well as visual electrophysiology testing deemed appropriate on an individual basis. A review was undertaken of paediatric case notes and of maternal antenatal urine toxicology.

Results: Ophthalmic abnormalities included reduced acuity (95%), nystagmus (70%), delayed visual maturation (50%), strabismus (30%), refractive errors (30%), and cerebral visual impairment (25%). Visual electrophysiology was abnormal in 60%. A quarter of the children had associated neurodevelopmental abnormalities. The majority of children with nystagmus (79%) had been treated for neonatal abstinence syndrome (NAS).

Conclusion:

Infants born to drug-misusing mothers prescribed methadone in pregnancy are at risk of a range of visual problems, the underlying causes of which are not clear. Those infants with NAS severe enough to receive pharmaceutical treatment may be at particular risk of developing nystagmus. The inclusion of visual electrophysiology in comprehensive visual assessment of children exposed to substance misuse in utero may help to clarify the underlying causes by differentiating abnormalities of retinal and cortical origin.

Introduction

Drug misuse in pregnancy is a significant problem which has both short and long term adverse consequences for the unborn child [START_REF] Burns | Methadone in pregnancy: treatment retention and neonatal outcomes[END_REF][START_REF] Dryden | Maternal methadone use in pregnancy: factors associated with the development of neonatal abstinence syndrome and implications for healthcare resources[END_REF][START_REF] Lloyd | Substance misuse research -neonatal abstinence syndrome. A new intervention: A community based, structured health visitor assessment[END_REF][START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] . Methadone, a synthetic opioid, has higher oral bio-availability than most opioids, long duration of action and less misuse potential [START_REF] Farid | The effects of maternally administered methadone, buprenorphine and naltrexone on offspring: review of human and animal data[END_REF] . It is commonly prescribed as a substitute for illicit opioids such as heroin, and is associated with more stable maternal lifestyle as well as reduced incidence of both intra-uterine growth restriction [START_REF] Hagopian | Neonatal outcome following following methadone exposure in utero[END_REF] and preterm birth 1;7 compared with non-treated heroin addicts. However, prescribed methadone is also associated with neonatal abstinence syndrome (NAS) in a majority of newborns, with symptoms severe enough to require pharmacological treatment in 45-80% of those 2;8 . The visual system is sensitive to adverse prenatal events, showing characteristic abnormalities after in utero exposure to alcohol and cocaine. A high incidence of strabismus has been associated with prenatal exposure to methadone [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] , and nystagmus in children born to drug-addicted mothers is being increasingly recognised 3;4 . Delayed visual development has also been reported 3;4 . Visual evoked potentials (VEPs) from adults receiving methadone were delayed relative to controls [START_REF] Bauer | Effects of chronic opioid dependence and HIV-1 infection on pattern shift visual evoked potentials[END_REF] . Electrophysiological data available so far from infants following in utero exposure to methadone show abnormal, smaller or slower VEPs after exposure relative to controls 10;11 . Three cases of older children (aged 4 years (N=2) and 12 years) reported normal flash VEPs [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] . We describe clinical, ophthalmological and visual electrophysiological findings in 20 children exposed to methadone and other drugs of misuse in utero.

Methods

This was a retrospective descriptive case series of children referred to a national paediatric visual electrophysiology service because of concerns regarding visual function and who had also been exposed in utero to methadone. Appropriate electrophysiological investigations were undertaken depending on the age of the child and presenting symptoms. Pattern-reversal VEPs were recorded to a black-and-white checkerboard at 100% contrast, mean luminance 60cdm -2 , subtending 30°×24° and presented at 1.1Hz. Pattern-onset VEPs were recorded to a black-and-white checkerboard at 100% contrast, interleaved with an isoluminant grey screen, subtending 30°×24° and presented at 1.1Hz. Flash VEPs were recorded to a hand-held diffuse flash with a time-integrated luminance of 11.7 cd s m -2 . In order to estimate acuity, step VEPs were recorded to black-and-white reversing checks using real-time analysis and a successive approximation algorithm to find spatial thresholds [START_REF] Mackay | Real time rapid acuity assessment using VEPs: development and validation of the 'step VEP' technique[END_REF] . All children also underwent ophthalmological and orthoptic review, often on several occasions, which included cover tests, ocular motility, cycloplegic refraction, and dilated fundoscopy. Visual acuity was assessed using age-appropriate tests; some subjects were unable to co-operate with behavioural acuity tests and step VEP acuity assessment was used instead. Delayed visual maturation was a retrospective diagnosis in babies with visual behaviour which was poorer than expected from their postnatal age but which improved by six months of age. Structured history taking was used for older children to seek evidence of cerebral visual impairment (CVI) [START_REF] Dutton | Dorsal stream dysfunction' and 'dorsal stream dysfunction plus': a potential classification for perceptual visual impairment in the context of cerebral visual impairment[END_REF] . Paediatric and neonatal case notes were reviewed, and details obtained regarding maternal antenatal urine toxicology when available. Subjects were excluded from the case series if they had been born before 32 weeks gestation or had another diagnosis which could potentially account for their visual abnormalities such as foetal alcohol syndrome or significant birth asphyxia.

Results

Twenty children met the inclusion criteria. All children had been exposed to prescribed substitute methadone in utero and a majority had also been exposed to illicit drugs, most commonly benzodiazepines (11/20, 55%) and heroin (8/20, 40%). Drug exposure as determined from case notes and urine toxicology is shown in Table 1. Twelve infants (60%) received pharmacological treatment for NAS. Age at first electrophysiology assessment ranged from 3 months to 7 years. Ocular and visual electrophysiological findings are shown in Table 2. The most common abnormalities demonstrated were reduced acuity (19/20, 95%), nystagmus (14/20, 70%), delayed visual maturation (10/20, 50%), strabismus (7/20, 35%) and refractive errors (6/20, 30%). Fundal examination was abnormal in two cases -one case had bilateral abnormal blood vessels crossing the macula which showed regression at five months of age, and the other had slight pallor of both optic discs. Five children (25%) had significant neurodevelopmental problems (developmental delay in four, cerebral palsy in one): three of these five (patients 2, 13 and 19) had CVI and one (patient 12) had a delayed flash VEP. CVI was found in a further two cases, giving an overall incidence of CVI of 5/20 (25%). Eleven of those 12 infants (92%) who had received pharmacological treatment for NAS demonstrated nystagmus, whilst only three of the eight infants (38%) who had not developed NAS severe enough to require treatment had nystagmus. This represents a significant difference (Fisher exact test, P=0.018) which was not evident for any of the other abnormalities. Nystagmus was horizontal in nature with the majority having a pendular wave-form although one patient exhibited a jerk type pattern. The observed characteristics of the nystagmus varied in being manifest, latent, or manifest with a latent component. Interestingly, patient 13 initially had manifest latent nystagmus which developed with time into latent nystagmus alone. Twelve patients (60%) had abnormal visual electrophysiology. One of these (patient 11) had a reduced amplitude cone and flicker ERG, but normal fundus and normal pattern-onset VEP to 60' checks. All other ERGs recorded were normal (N=13). Flash VEPs were recorded from 11 patients, and were normal in eight (73%) and delayed in three cases (patients 5, 10 and 12 (Figure 1A)). An abnormal flash VEP was associated with a history of NAS and with nystagmus. Pattern-reversal VEPs were recorded from six patients and were normal in two cases but delayed or absent in four cases (patients 5, 9 (Figure 1B), 17 (Figure 1C) and 20). Pattern-onset VEPs were recorded in six cases, and were normal in five (e.g. Figure 1D, patient 17) but delayed in one (patient 5). Eleven subjects had their visual acuity estimated using the step VEP; in nine cases (82%), acuity was abnormal for age. These acuity estimates agreed with contemporary behavioural acuity assessments in all nine cases where both were available. Discussion Despite increasing awareness of risks to the unborn child from substance misuse in pregnancy, the incidence of maternal drug misuse is rising: anonymous screening in a UK clinic suggested that 11-16% of expectant women use at least one illicit substance during pregnancy [START_REF] Sherwood | Substance misuse in early pregnancy and relationship to fetal outcome[END_REF] .

The most commonly misused substances in the UK are opiates, although the use of cocaine is increasing. There are an estimated 250,000 to 350,000 children of problem drug users in the UK alone [START_REF] Harm | The report of the Inquiry by the Advisory Council on the Misuse of Drugs[END_REF] , who represent a substantial burden to health, social and educational services 2 . As with Mulvihill et al (2007) [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] , our report was prompted by an awareness of a history of in utero methadone exposure in an increasing number of patients referred because of visual problems. A high proportion (11/14, 79%) of the children who presented with nystagmus in this case series had significant NAS. This is higher than reported elsewhere (7/14 (50%) [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] ), but supports the overall picture of an association of nystagmus with NAS. Nystagmus has a higher incidence (3/72 (4%)) [START_REF] Lloyd | Substance misuse research -neonatal abstinence syndrome. A new intervention: A community based, structured health visitor assessment[END_REF] in the general drug-exposed population (i.e. not children referred because of eye problems) than in the population at large (24 per 10,000) [START_REF] Sarvananthan | The prevalence of nystagmus: The Leicestershire nystagmus survey[END_REF] .

It has been proposed that infantile nystagmus could be caused by visual deprivation, resulting in oculomotor development outpacing sensory development [START_REF] Harris | A developmental model of nystagmus[END_REF] , and it is certainly possible that the children reported here may have experienced sensory delay or deficit as infants. An alternative explanation is abnormal µ-opioid receptor binding in the developing brainstem [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] . Steadiness of horizontal gaze is governed by a neural integrator which depends heavily on the nucleus prepositus hypoglossi and medial vestibular nucleus (MVN) [START_REF] Cannon | Loss of the neural integrator of the oculomotor system from brain stem lesions in monkey[END_REF] . The MVN contains µ-opioid receptors [START_REF] Lin | Direct excitatory opiate effects mediated by non-synaptic actions on rat medial vestibular neurons[END_REF] , which are activated by opiates and by methadone. During development, opioid receptor activation inhibits proliferation and neuronal differentiation [START_REF] Hauser | Opioids intrisically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: differential impact of mu and delta receptor activation on proliferation and neurite elongation[END_REF] while exposure to morphine in utero restricts cell proliferation and maturation [START_REF] Seatriz | Effects of opiates on neuronal development in the rat cerebral cortex[END_REF] . It is possible that abnormal receptor binding in the developing neural integrator could provide a mechanism for nystagmus to develop. An association has been suggested between additional benzodiazepine exposure in utero and nystagmus [START_REF] Mulvihill | Nystagmus secondary to drug exposure in utero[END_REF] , but this was not found in our case series (Fisher exact test, p=0.62). Disruption of coordinated binocular vision early in life leads to strabismus and varying degrees of amblyopia, with the extent of impairment dependent on the onset time, duration and type of visual deprivation [START_REF] Mustari | How disturbed visual processing early in life leads to disorders of gaze-holding and smooth pursuit[END_REF] . One third of children in the current study had strabismus, much higher than in a similarly-aged general population (1.3%) [START_REF] Friedman | Ophthalmic screening of 38,000 children, age 1 to 2 1/2 years, in child welfare clinics[END_REF] . The proportions of children in the current study with strabismus who had significant NAS and who had no NAS are not statistically different (4/12 (33%) versus 3/8 (37%), Fisher exact test, p=1). Abnormal visual electrophysiology was a frequent finding. Reduced step VEP acuity was consistent with reduced acuity from contemporary behavioural acuity assessments. The VEP is a cortically generated response to stimulation of the central visual field; prolonged latencies and reduced amplitudes are associated with neural demyelination and axonal loss respectively. Delayed or absent peaks of flash and pattern VEPs were noted in this study, consistent with findings noted in the neonatal period [START_REF] Mcglone | Visual evoked potentials in infants exposed to methadone in utero[END_REF] ; the latter study suggests a pre-natal cause. Pattern-reversal VEPs were abnormal in 4/6 cases while pattern-onset VEPs were abnormal in only 1/6 cases, consistent with the presence of nystagmus. However, one patient (5) had delayed pattern-reversal VEPs despite absence of nystagmus. It is possible that the pattern VEP delay is related to methadone exposure 11 : both acute and maintenance methadone treatment delay the pattern-reversal VEP in adults 9;11;24 . Opioid signalling plays a role in myelination and axonal growth, which could affect VEP latency [START_REF] Sanchez | Opioid addiction and pregnancy: perinatal exposure to buprenorphine affects myelination in the developing brain[END_REF] . There were very few abnormal fundi findings suggesting that posterior visual pathway dysfunction is important. Cerebral visual impairment was diagnosed in 25% of these children, but this is likely to be a substantial underestimate as CVI is commonly diagnosed around school entry age. This entire cohort had been exposed to methadone in utero, but also to the many associated adversities, including physical and mental ill health, poor nutritional status 1 and excessive alcohol consumption [START_REF] Burns | Methadone in pregnancy: treatment retention and neonatal outcomes[END_REF] . We cannot be certain whether the visual abnormalities described are secondary to substitute methadone, ongoing illicit maternal opiate and/or benzodiazepine misuse, pharmacological agents (including oral morphine solution and/or phenobarbitone) used to treat NAS, or other as yet unidentified factors. The difficulties inherent in following children of vulnerable families means that it is likely that this group of infants is representative of a larger population of methadone-exposed children who have similar but undiagnosed ophthalmic problems.

Conclusion

Infants born to drug-misusing mothers prescribed methadone in pregnancy are at risk of a range of visual problems, the underlying causes of which are not clear. Those infants who receive treatment for NAS may be at particular risk of developing nystagmus. Abnormalities occur at all stages of the visual pathway, and the inclusion of visual electrophysiology in comprehensive visual assessment of children exposed to substance misuse in utero may help to clarify the underlying causes by differentiating abnormalities of retinal and cortical origin. Controlled studies are necessary to delineate the effects of in utero drug exposure on visual outcomes.

Figure 1 :

 1 Figure 1: Examples of VEPs from three patients. A: flash VEP from subject 12, showing small, delayed peaks. B: Pattern reversal VEP (60' checks) from subject 9, showing small, delayed peak. C: absent pattern reversal VEP (60' checks) from subject 17; in contrast, D shows normal patternonset (60' checks) from the same subject. In all frames, control recordings from age-matched normal infants or children are shown in grey below the patients' trace.

Table 1 :

 1 Drug exposure and systemic findings

	patient no.	gestation (weeks) methadone heroin BDZ	other drugs	alcohol treatment for NAS	other diagnoses	brain scans
	1	38
	2	32			preterm, developmental delay
	3	39
	4	40	cannabis
	5	38				MRI: abnormal white matter
	6	39
	7	40			mild cerebral palsy
	8	38	cocaine			CT normal
	9	36	cocaine			MRI normal
	10	34	cannabis		preterm
	11	40	cannabis
	12	35	cannabis		developmental delay, dysmorphic
	13	40			developmental delay
	14	38
	15	36				MRI normal
	16	38	antidepressants (not specified)
	17	41
	18	40	cannabis
	19	37			developmental delay	MRI normal
	20	39	dihydrocodeine

BDZ: benzodiazepines, NAS: neonatal abstinence syndrome, MRI: magnetic resonance imaging, CT computerised tomography.

Table 2 :

 2 Ocular and electrophysiology findings VA: visual acuity, f & f: fixing and following, BEO: both eyes open, DVM: delayed visual maturation, ET: esotropia, XT: exotropia, MT: microtropia, M: myopia, H: hypermetropia, A: astigmatism, CVI: cerebral visual impairment, ERG: electroretinogram, VEP: visual evoked potential. *VA in Snellen equivalent. For visual acuity, the age at test in months is given in brackets next to test findings.

	patient no.	VA at presentation (age, months)	most recent VA (age, months) DVM nystagmus strabismus refractive error	fundus	CVI ERG flash VEP	pattern reversal VEP	pattern onset VEP	step VEP acuity
	1 momentary eye contact (3) 6/19, 6/19 (20)	XT		normal	normal normal		raised
	2	6/12 BEO (14)	6/19, 6/15 (28)			normal		normal	raised
	3	6/76 BEO (13)				normal	normal
	4	6/48 BEO (6)	6/9.5 BEO (11)			normal			normal	raised
	5	6/38 BEO (7)	6/24 * BEO (25)			normal	normal delayed & small	abnormal	abnormal
	6	6/130 BEO (7)	6/9.5 BEO (20)			normal	normal normal		normal
	7	6/19, 6/12 (63)		ET		normal	normal		normal
	8		6/18 *, 6/15 * (48)			normal	normal normal		raised
	9	6/76 BEO (6)	5/6, 5/6 (51)		HA	normal	normal normal	absent	raised
	10	6/30 by VEP (7)		NK		normal	delayed		normal	raised
	11	6/24 BEO (13)	6/24 BEO (18)			normal	small		normal
	12	6/500 BEO (3)	6/120, 6/9.5 (35)	XT	MA (right eye only)	vessels across macula	normal delayed		raised
	13	Not f & f (2)	6/6, 6/6 (68)	possible MT		normal		normal
	14	6/500 BEO (3)	6/9.5, 6/15 (46)	XT	H	normal			raised
	15	f & f (5)	6/12 *, 6/12 * (60)			normal	normal normal
	16	6/120 BEO (8)	3/18, 3/18, 3/12 BEO (45)		HA	normal	normal normal		raised
	17	f but not f (3)	6/12 BEO (22)			normal	normal	absent	normal
	18	6/19, 6/15 * (71)				normal	normal
	19	6/24 BEO (27)	6/36 BEO (37)	ET	HA	slight disc pallor	normal normal
	20	6/60, 6/13 (77)			A	normal	normal	delayed	raised

Acknowledgements: We thank Professor Gordon Dutton for helpful comments on the manuscript. LMcG was funded by the Yorkhill Children's Foundation.

Competing interest: none declared. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence.


Muhammad Amer Awan

Fiona Roberts

Bernie Hegarty

Kanna Ramaesh

The outcome of deep anterior lamellar keratoplasty in HSV related corneal scarring, complications and graft survival

Keywords: herpes simplex, keratitis, corneal scarring, lamellar keratoplasty, rejection

Aims:

To determine the visual outcome, graft survival and complications after deep anterior lamellar keratoplasty (DALK) in patients with herpes simplex virus (HSV) related corneal scarring.

Methods:

A retrospective analysis of the patients who had DALK for HSV related corneal scarring between January 2004 and February 2007 was performed. Mean follow up was 30 months (Range: 16 to 48 months). The statistical significance of host corneal vascularisation was determined using Fisher's exact test.

Results:

There were 18 eyes from 18 patients and the mean age was 57 years. Preoperative VA ranged from HM to 6/12. Fifty percent of the eyes achieved VA of 6/12 or better postoperatively. Six eyes (33%) had recurrence of HSV related inflammation, eight eyes (including four eyes with recurrence of HSV related inflammation) developed graft rejection and four eyes (including two eyes with recurrence of HSV related inflammation) had bacterial keratitis. The graft survival rate was 83%. Three eyes developed glaucoma and one eye required trabeculectomy. Immunohistochemistry (IHC) revealed that HSV was focally positive or equivocal in four recipient corneal buttons and transmission electron microscopy (TEM) showed intracellular HSV virions in two of them.

Conclusions:

This is the largest series of DALK for herpetic corneal scarring which shows a comparable visual outcome and better graft survival rate than penetrating keratoplasty. There is significant risk of recurrence of HSV related inflammation and graft rejection which requires timely recognition and adequate management.

Introduction:

Herpes simplex keratitis (HSK) is one of the major causes of visual morbidity. Corneal scarring occurs in 18-28% with reduction in visual acuity to less than 6/12 in 10-25%. [START_REF] Liesegang | Epidemiology of ocular herpes simplex: natural history in Rochester, Minn, 1950 through 1982[END_REF] Despite developments in surgical and therapeutic approaches to date, corneal graft survival in the patients with HSK is significantly lower than in the patients with non-herpetic conditions. Patients undergoing corneal graft for sequelae of herpes simplex virus (HSV) keratitis are at higher risk for adverse corneal allograft outcomes when compared with individuals undergoing grafting for conditions such as keratoconus and Fuchs' corneal dystrophy. [START_REF] Epstein | Penetrating keratoplasty for herpes simplex keratitis and keratoconus. Allograft rejection and survival[END_REF][START_REF] Thompson Rw | Long-term graft survival after penetrating keratoplasty[END_REF] The postoperative course can be complicated by high rates of HSV recurrence, graft rejection, and graft failure. [START_REF] Cohen | Corneal transplantation for herpes simplex keratitis[END_REF][START_REF] Foster | Penetrating keratoplasty for herpes simplex keratitis[END_REF][START_REF] Lomholt | Recurrence and rejection rates following corneal transplantation for herpes simplex keratitis[END_REF][START_REF] Ficker | Long term prognosis for corneal grafting in herpes simplex keratitis[END_REF] The purpose of study was to determine the visual outcome, graft survival and complications after DALK in eyes with HSV related corneal scarring.

Materials & methods:

A retrospective, non-comparative case review of patients with HSV related corneal scarring who had DALK between January 2004 and February 2007 was undertaken. All the patients had inactive disease (at least 2 years) at the time of allograft and the indication of corneal graft was HSV related corneal scarring. Two eyes had corneal perforation previously; one treated with gluing and other required SFCAP (superior forniceal conjunctival advancement pedicle). All patients had primary corneal graft and DALK performed by a single surgeon (KR) by using Anwar's big bubble technique. [START_REF] Anwar | Big-bubble technique to bare Descemet's membrane in anterior lamellar keratoplasty[END_REF] Twelve to sixteen interrupted 10/0 nylon sutures were used to suture the graft depending on size of the graft.

Routine haematoxylin and eosin (H&E) histology, standard immunohistochemistry (IHC) for HSV and transmission electron microscopy (TEM) were also preformed in the recipient corneal buttons. The postoperative treatment regime comprised oral acyclovir 400mg BD for 12 months, chloramphenicol 0.5% and dexamethasone 0.1% eye drops QID for 4 weeks. Topical steroids were gradually reduced in strength and frequency over 6-9 months. All the patients were closely monitored for signs of inflammation, recurrence of HSK, graft rejection, infection, suture-related problems, raised intraocular pressure, and refractive outcome. Recurrence of HSK was described as epithelial, stromal or endothelial disease. Episodes of graft rejection were diagnosed in the presence of decreased vision, stromal oedema with or without subepithelial infiltrates, and anterior segment activity. Mean follow up was 30 months (Range: 16 to 48 months). The statistical significance of host corneal vascularisation and its influence on recurrence of HSK, graft rejection and failure was determined by using Fisher's exact test.

Results:

The total no of the patients was 18 which included 11 females and 7 males. The mean age was 57 years (range 20 to 75 years). Preoperative best corrected visual acuity (BCVA) varied from HM to 6/12 and only one patient had VA of 6/12. Vascularisation of the host cornea was observed in 12 eyes (7 eyes had both superficial and deep vessels). The average donor graft size was 8 mm (range 7.5 to 9.5 mm).

Microperforation of Descemet's membrane was observed in 4 eyes during stromal dissection phase but this did not affect the subsequent surgical steps leading to the uneventful completion of DALK procedure. At the end of operation 20% SF6 was injected intracamerally to prevent formation of double AC (anterior chamber). Postoperatively one eye developed double AC that required intracameral injection of SF6 gas on day 3. All patients had removal of graft sutures at an average time interval of 9 months.

Postoperatively nine patients (50%) achieved VA of 6/12 or better and 83% of the patients 6/60 or better [Table 1]. Mean postoperative astigmatism was 5.6 ± 4.4 dioptres (D) and mean spherical equivalent (SEQ) was -1.5± 2.2 D. Nine eyes had postoperative BCVA worse than 6/12; graft failure leading to opaque vascularised grafts was the causative factor in 3 eyes and interface scarring was present in 2 eyes. Other causes of poor postoperative visual outcome were glaucoma (2 eyes), stromal scarring due to bacterial keratitis (1 eye) and marked astigmatism (1 eye).

There was no statistical significant relationship between host corneal vascularisation and recurrence of HSK (p value = 0.4), graft rejection (p value = 0.2) and failure (p value = 0.5). Six eyes (33%) had recurrence of HSV related inflammation (five eyes with epithelial disease & one with keratouveitis) and were managed with topical acyclovir and steroids [Figure 1]. Four eyes (22%) presented with bacterial keratitis that included the two eyes with HSV recurrence and were treated with intensive use of topical 5% cefuroxime and 1.5% gentamicin [Figure 1]. Allograft rejection occurred in eight eyes (44%) including the four eyes with recurrence of HSV related inflammation and was managed with topical steroids, oral steroids and intravenous methyl prednisolone [Figure 2]. Our graft survival rate was 83% after 3 years. Three eyes developed glaucoma and one eye required trabeculectomy. Histology in each case showed features in keeping with scarring with associated vascularisation in some cases. IHC of the recipient corneal buttons showed focal positivity or equivocal results in four recipient corneal buttons whereas TEM revealed intracellular HSV virions in two of these recipient corneal buttons [Figure 3 &4]. To our knowledge this is the largest case series of patients who had DALK for HSV related corneal scarring. It is often difficult to determine whether a vascular or avascular corneal scar is herpetic in origin. The diagnosis of previous HSK is particularly important for patients undergoing corneal graft both for prognosis and requirement for post-surgical antiviral therapy. Lamellar keratoplasty has been performed to treat a wide variety of corneal pathologies without endothelial involvement. However, the technique is difficult, and DM perforation is a well-described complication of DALK surgery. There are different studies showing DM perforation rate varying from 4% to 39%. [START_REF] Sugita | Deep lamellar keratoplasty with complete removal of pathological stroma for vision improvement[END_REF][START_REF] Watson | Comparison of deep lamellar and penetrating keratoplasty in patients with keratoconus[END_REF][START_REF] Noble | Deep Anterior Lamellar Keratoplasty (DALK): visual outcome and complications for a heterogeneous group of corneal pathologies[END_REF] Our study showed DM perforation in 22% of the eyes but none of them required conversion to PK.

There is increasing evidence that reactivation of latent HSK plays an important role in graft failure causing a high rate of allograft rejection. [START_REF] Kaye | Human herpesviruses in the cornea[END_REF] Primary allograft rejection after PK for HSK was observed in 29% of the grafts in the first year and in 46% during the first 2 years. [START_REF] Lomholt | Recurrence and rejection rates following corneal transplantation for herpes simplex keratitis[END_REF] Studies have revealed that graft rejection is an infrequent complication of DALK surgery and is seen in 0% to 10% of the eyes undergoing surgery for heterogeneous corneal problems. [START_REF] Watson | Comparison of deep lamellar and penetrating keratoplasty in patients with keratoconus[END_REF][START_REF] Noble | Deep Anterior Lamellar Keratoplasty (DALK): visual outcome and complications for a heterogeneous group of corneal pathologies[END_REF][START_REF] Timarchi | Deep lamellar keratoplasty[END_REF] Our study showed 3 year graft rejection rate of 33% which is better than PK and it is comparable to the graft rejection rate in DALK for herpetic corneal scarring as described by Noble et al. [START_REF] Noble | Deep Anterior Lamellar Keratoplasty (DALK): visual outcome and complications for a heterogeneous group of corneal pathologies[END_REF] In our series all eyes with graft rejection except one had preoperative host bed corneal vascularisation. We could not find any statistically significant influence of corneal vascularisation on graft rejection or HSV related inflammation. Currently, it is also unclear whether risk factors such as corneal vascularisation, previous rejection episodes, and large grafts are associated with a higher rejection rate in patients undergoing DALK, as is the case with PK.

The incidence of recurrence of HSK after penetrating keratoplasty (PK) has been reported to 25% in first year and 44% in first two years, [START_REF] Lomholt | Recurrence and rejection rates following corneal transplantation for herpes simplex keratitis[END_REF] and is a major cause of corneal graft failure in the patients. In our series the recurrence of HSK was 5% in first year which is significantly less in comparison to previous studies. [START_REF] Lomholt | Recurrence and rejection rates following corneal transplantation for herpes simplex keratitis[END_REF] The majority of the patients had recurrence in the second year. Our patients had prophylactic oral acyclovir for 12 months which may have reduced the recurrence of HSK. Van Rooij et al also demonstrated in their randomized controlled trial (RCT) that oral acyclovir effectively prevents herpesrelated recurrences after penetrating keratoplasty in herpetic eye disease. [START_REF] Van Rooij | A retrospective study on the effectiveness of oral acyclovir to prevent herpes simplex recurrence in corneal grafts[END_REF] In 50% of the eyes we achieved VA of 6/12 or better, 83% obtained 6/60 or better and 3 eyes with graft failure had VA worse than 6/60. The mean SEQ was -1.5 D which is almost similar to the other series of DALK patients, with reported values between -1.65 and -4.13 D. [START_REF] Watson | Comparison of deep lamellar and penetrating keratoplasty in patients with keratoconus[END_REF][START_REF] Funnell | Comparative cohort study of the outcomes of deep lamellar keratoplasty and penetrating keratoplasty for keratoconus[END_REF][START_REF] Coombes | Deep lamellar keratoplasty with lyophilised tissue in the management of keratoconus[END_REF] The main causes of poor visual outcome were graft failure, interface and stromal scarring and glaucoma. In our series 3 years graft survival rate was 83% which is higher than 2 year survival rate of 67% as reported by Lomholt et al [START_REF] Lomholt | Recurrence and rejection rates following corneal transplantation for herpes simplex keratitis[END_REF] in their series of PK in herpetic corneal scarring. This may be related to DALK procedure as there is preservation of host corneal endothelium and no risk of endothelial rejection. All three eyes with graft failure had vascularisation of the host corneal bed and one of the grafts was large (9.5mm). One of them previously had corneal perforation and other eye had pre existing glaucoma. Postoperatively one eye developed bacterial corneal abscess and stromal rejection; and the second eye had recurrence of HSV related keratouveitis, two episodes of stromal rejection and uncontrolled glaucoma which required trabeculectomy within the first 2 years. The third eye had bacterial keratitis that eventually led to graft failure.

Although, there are no studies directly comparing the duration of existence of HSV-1 in the trigeminal ganglion and cornea, the available evidence suggests that detection of the virus in the cornea may be time dependent. Kaye and colleagues 12 demonstrated that HSV-1 DNA was detected significantly more frequently in the corneas of patients who had their last documented episode of HSK within 2 years of surgery. The longer the period between the last episode of HSK and surgery, the less likely it is that virus will be detected. There are different techniques which may be used to detect HSV but we used IHC and TEM. IHC has a reasonably good sensitivity and specificity for the detection of HSV-1 (74% and 85%, respectively). [START_REF] Kaye | Human herpesviruses in the cornea[END_REF] PCR was not performed in our study as the corneal specimens were formalin fixed and embedded in paraffin for more than 6 months and length of time in paraffin has been shown to critically influence PCR results. [START_REF] Ben-Ezra | Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction[END_REF] Our series demonstrated focal positivity or equivocal results in four recipient corneas on IHC and two of them also showed intracellular HSV virions on TEM. All patients had last episode of HSV keratitis more than 2 years from the time of surgery which may depict that detection is time dependent.

In summary this is the largest series of DALK in HSV related corneal scarring that reveals comparable visual outcome and better graft survival rate than PK. There remains a significant risk of recurrence of HSV related inflammation and graft rejection which requires timely recognition and adequate management.

Legends:

  corrected visual acuity, S = Superficial, D = Deep, CE = Cataract extraction, AC = Atopic conjunctivitis, AK = Astigmatic keratotomy, CS = Compressive suture, SFCAP = Superior forniceal conjunctival advancement pedicle, PXF = Pseudo exfoliation, RK= Radial keratotomy Discussion:

Figure 1 :Figure 2 :

 12 Figure 1: The distribution of HSV related inflammation and bacterial keratitis episodes depending on their year of presentation

Figure 3 :

 3 Figure 3: Transmission electron microscopy of the recipient corneal button showing occasional intranuclear HSV virions (arrows).

Figure 4 :

 4 Figure 4: Immunohistochemistry of the recipient corneal button illustrating focal positive staining of stromal keratocytes (arrows).

Figure 1 :Figure 2 :

 12 Figure 1: The distribution of HSV related inflammation and bacterial keratitis episodes depending on their year of presentation

Figure 3 :

 3 Figure 3: Transmission electron microscopy of the recipient corneal button showing occasional intranuclear HSV virions (arrows).

Figure 4 :

 4 Figure 4: Immunohistochemistry of the recipient corneal button illustrating focal positive staining of stromal keratocytes (arrows).

Table 1 : Description of preoperative & postoperative visual acuities, ocular co-morbidities, complications, immunohistochemistry (IHC), transmission electron microscopy (TEM) and any further surgical procedures.

 1

	Pts	Pre-op	Ocular co-	Vascularisation	Post-op	Recurrence	Rejection	Bacterial	Failure	IHC	EM	Additional
		BCVA	morbidities	of host cornea	BCVA	of HSK	(type)	infection				surgical
					(month)	(type)						procedures
	1	6/18	N	N	6/9 (12)	N	N	N	N	-ve	-ve
	2	CF	Dry eyes,	Y (S & D in 1	6/24 (14) N	N	N	N	-ve	-ve	CE
			AC,	quadrant)
			glaucoma
	3	HM	Dry eyes	Y (S & D in 1	CF (15)	N	N	Y	Y	-ve	-ve	SFCAP,
				quadrant)								Botox
												ptosis
	4	1/60	N	Y (S & D in 3	6/12 (16) N	N	N	N	-ve	-ve
				quadrants)
	5	6/12	PXF	N	6/6 (38)	N	Y	N	N	-ve	-ve	AK, CS,
							(Epithelial)					CE
	6	CF	N	Y (S & D in 1	6/6 (48)	Y	Y	N	N	-ve	-ve
				quadrant)		(Epithelial)	(Epithelial)
	7	6/36	N	Y (D in 1	6/36 (22) Y	Y	Y	N	-ve	-ve
				quadrant)		(Epithelial)	(Stromal)
	8	6/24	N	N	6/36 (18) N	N	N	N	-ve	-ve
	9	6/36	N	Y (D in 1	CF (28)	Y	Y (Stromal,	N	Y	-ve	-ve	CE, Trab
				quadrant)		(Keratouveit	twice)
						is)
	10	6/36	N	Y (S & D in 1	6/12 (21) N	Y	N	N	Equivocal	+ve
				quadrant)			(Stromal)
	11	HM	N	N	6/12 (22) N	N		N	Equivocal	+ve
	12	6/18	N	N	6/9 (28)	Y	N	N	N	Focal	-ve
						(Epithelial)				positivity
	13	3/36	N	Y (S & D in 2	2/60 (21) N	Y	Y	Y	Focal	-ve
				quadrants)			(Stromal)			positivity
	14	HM	N	N	6/18 (47) N	N	N	N	-ve	-ve	CE
	15	1/60	N	Y (S in 1	6/9 (45)	N	N	N	N	-ve	-ve	AK, CS,
				quadrant)		(Epithelial)						CE
	16	1/60	N	Y (D in 1	6/60 (12) Y	Y	Y	N	-ve	-ve
				quadrant)		(Epithelial,	(Stromal)
						twice)
	17	1/60	N	Y (S & D in 1	6/18 (30) Y	N	N	N	-ve	-ve	CE, CS,
				quadrant)		(Epithelial)						AK, RK
	18	1/60	N	Y (S in 1	6/12 (47) N	Y
				quadrant)			(Stromal)


Dr Katrin Petermeier
email: katrin.petermeier@med.uni-tuebingen.de

MD Charlotte Frank

Florian Gekeler

MD Martin S Spitzer

MD Andre Messias

MD Peter Szurman

Influence of the pupil size on visual quality and spherical aberration after implantation of the Tecnis ® 1-piece

Keywords: aspheric IOL; contrast sensitivity, depth of focus, spherical aberration, pupil size

Purpose: To evaluate the influence of the pupil size on clinical results and objective parameters of optical quality of the Tecnis ® ZCB00.

Introduction

With refinements in surgical technique, intraocular lens design and biometry modern cataract surgery has advanced beyond mere restoration of visual acuity. After the introduction of the Tecnis ® Z9000 in 2001 with a negative spherical aberration, the attention of cataract surgeons has been directed to optical aberrations, contrast sensitivity and visual quality.

Aspheric IOLs compensate the positive spherical aberration of the cornea like the crystalline lens does in the young eye. They compensate the corneal spherical aberration even better than the natural lens in elderly people and thus lead to a total spherical aberration like in a young eye [START_REF] Rekas | Optical quality in eyes with aspheric intraocular lenses and in younger and older adult phakic eyes: Comparative study[END_REF][START_REF] Rekas | Comparison of higher order aberrations with spherical and aspheric IOLs compared to normal phakic eyes[END_REF].

In a recent review Montés-Micó et al. report on the advantages of aspheric IOLs like reduction of the total spherical aberration and improved contrast sensitivity [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF]. But the pupil size is a crucial criterion for the comparison of functional results of spherical and aspheric lenses [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF][START_REF] Awwad | Contrast sensitivity and higher order aberrations in eyes implanted with AcrySof IQ SN60WF and AcrySof SN60AT intraocular lenses[END_REF][START_REF] Denoyer | Quality of vision after cataract surgery after Tecnis Z9000 intraocular lens implantation; effect of contrast sensitivity and wavefront aberration improvements on the quality of daily vision[END_REF][START_REF] Kohnen | Aspheric intraocular lenses[END_REF][START_REF] Mester | Comparison of the AcrySof IQ aspheric blue light filter and the Acrysof SA60AT intraocular lenses[END_REF][START_REF] Yamaguchi | Effect of spherical aberration on visual function under photopic and mesopic conditions after cataract surgery[END_REF]. Most studies report that the reduction of total spherical aberration in eyes with aspheric IOLs compared to eyes with spherical IOLs is more evident at large pupil sizes [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF][START_REF] Awwad | Contrast sensitivity and higher order aberrations in eyes implanted with AcrySof IQ SN60WF and AcrySof SN60AT intraocular lenses[END_REF][START_REF] Denoyer | Quality of vision after cataract surgery after Tecnis Z9000 intraocular lens implantation; effect of contrast sensitivity and wavefront aberration improvements on the quality of daily vision[END_REF][START_REF] Kohnen | Aspheric intraocular lenses[END_REF][START_REF] Mester | Comparison of the AcrySof IQ aspheric blue light filter and the Acrysof SA60AT intraocular lenses[END_REF][START_REF] Yamaguchi | Effect of spherical aberration on visual function under photopic and mesopic conditions after cataract surgery[END_REF][START_REF] Bellucci | Comparison of wavefront aberrations and optical quality of eyes implanted with five different intraocular lenses[END_REF]. Bellucci et al. could show that the reduction of spherical aberration leads to a refractive effect towards myopia at large optical zones [START_REF] Bellucci | Comparison of wavefront aberrations and optical quality of eyes implanted with five different intraocular lenses[END_REF]. Denoyer et al. found a change in refraction towards myopia at large pupil sizes [START_REF] Denoyer | Quality of vision after cataract surgery after Tecnis Z9000 intraocular lens implantation; effect of contrast sensitivity and wavefront aberration improvements on the quality of daily vision[END_REF]. Mrochen et al. showed that even in young phakic eyes the critical pupil size, at which the optical aberrations of the eye deteriorate the quality of the retinal image, is 3.3 mm for 50% and 4.5 mm for 89% of the eyes [START_REF] Mrochen | Fundamentals of wavefront-guided refractive corneal surgery[END_REF]. Finally, we know from clinical experience that medicamentous pupil dilation in older or pseudophakic patients leads to a blurred vision.

Until now the influence of the pupil size on quality of vision with an aspheric IOL has not been explicitly evaluated yet. In our study we evaluated this influence after implantation of the new Tecnis ® ZCB00 IOL.

Patients and Methods

The presented study included 51 eyes of 51 consecutive patients who underwent phakoemulsification and injector implantation of a Tecnis ® 1-piece (ZCB00) intraocular lens for cataract. This IOL is a onepiece IOL made from hydrophobic acrylic material with a modified anterior surface and a negative spherical aberration of -0.27 µm at the 6 mm optical zone. Exclusion criteria were ocular disease other than cataract, previous ocular surgery and inflammation. Preoperative data included full ophthalmological examination and assessment of best-corrected visual acuity. Intraocular lens calculation was performed using an IOLMaster (Carl Zeiss Meditec AG, Jena, Germany) applying the SRK-T (A=119.4) formula.

Phakoemulsification was performed through a 2.75 mm temporal clear cornea tunnel incision with a capsulorrhexis of 5-5.5 mm and in-the-bag injector implantation of the Tecnis ® 1-piece intraocular lens.

Postoperatively, patients were evaluated for both uncorrected and best corrected visual acuity using ETDRS charts at a distance of 4 m. Corneal topography was performed with the C-scan (Technomed, Baesweiler, Germany) and Corneal spherical aberration was calculated for 3, 4, 5 and 6 mm pupil sizes.

Wavefront analysis was performed with dilated pupils with the Ocular Wavefront Analyzer (Schwind, Kleinostheim, Germany). This instrument measures total wavefront aberrations of the eye using the Hartmann-Shack principle. In order to evaluate not only the objective refraction but also the spherical aberration of the eye, the sphere, cylinder and the Zernike term Z4.0 was analyzed. Five measurements were obtained with a standard deviation not exceeding 0.05 μm. Every measurement was then evaluated for a 3, 4, 5, and 6 mm optical zone. The spherical equivalent of each eye was calculates by adding half the cylinder to the sphere (sphere + cylinder/2).

After medicamentous pupil dilation contrast sensitivity was tested monocularly with F.A.C.T-Charts (Ginsburg CST-1500, Vision Science Research Corporation, San Ramon, CA) under photopic (85 cd/m 2 ) and mesopic (6 cd/m 2 ) luminance levels at a spatial frequency of 6 cycles/degree. The defocus profiles (visual acuity over imposed defocus) were registered by measuring monocular visual acuity at a distance of 4 m starting from best distance correction and then defocusing with added lenses in 0,25 dioptre steps from -1.5 D to +1.5 D. For statistical calculation, visual acuity was converted into logMAR. To simulate different pupil sizes, both the contrast sensitivity and the defocus profile were evaluated with a pinhole of 3 mm and 5 mm. Statistical analysis was performed using the paired T-Test. P < 0.05 was considered to indicate a statistically significant difference.

Results

The mean follow-up period was 3.0 ± 0.4 months. Patients' mean age was 71.0 ± 8.1 years (range 59 -92). Mean intraocular lens power was 22.0 ± 3.0 D. (range 14.0-29.0). Monocular uncorrected visual acuity was 20/25 ± 8 letters (LogMar 0.1 ± 0.16), bestcorrected visual acuity was 20/18 ± 4 letters (LogMar -0.04 ± 0.08). The postoperative residual refractive error was -0.18 ± 0.5 D.

The residual refractive error (spherical equivalent) as measured with the wavefront analyzer was -0.5 ± 0.63 D, -0.53 ± 0.59 D, -0.53 ± 0.64 D and -0.56 ± 0.71 D for 3, 4, 5 and 6 mm optical zone, respectively. There was no statistically significant difference (figure 1).

In 8 eyes the corneal spherical aberration could not be evaluated for the 6 mm optical zone, so that these eyes were excluded from evaluation both for the corneal and the total spherical aberration. The corneal spherical aberration was 0.02 ± 0.01 µm, 0.06 ± 0.03 µm t, 0.14 ± 0.09 µm and 0.27 ± 0.22 µm for 3, 4, 5 and 6 mm optical zone, respectively. Statistical analysis (paired T-Test) revealed a statistically significant difference between the spherical aberration of the 3 and 4 mm, 4 and 5 mm, 5 and 6 mm optical zone (P < 0.05) (figure 2). In contrast, the total spherical aberration of the different optical zones was not statistically significantly different: -0.01 ± 0.02 µm, 0.0 ± 0.03 µm, 0.0 ± 0.06 µm and 0.0 ± 0.08 µm, for 3, 4, 5 and 6 mm optical zone, respectively (figure 2). The total spherical aberration and the corneal aberration were highly significantly different for all optical zones (P < 0.001).

Contrast sensitivity under mesopic and photopic conditions with a simulated pupil size of 3 and 5 mm is shown in figure 3. The contrast sensitivity with a 5 mm pupil size remains at the same level as with a 3 mm pupil size under mesopic and photopic conditions (P=0.91 and 0.37 respectively).

The mean monocular defocus profiles with a 3 and a 5 mm simulated pupil size are shown in figure 4. In none of the 52 eyes, the defocus curves were statistically significantly different, nor were the mean values of all eyes (P=0.19). The best-corrected visual acuity was 20/18 ±

Discussion

We have shown that the aspheric design of the Tecnis ® 1-piece compensates the positive corneal spherical aberration at all pupil sizes and thus leads to a high optical quality not only at a pupil size of 3 mm, but also at 5 mm.

Visual acuity and refraction

In our study we found no objective change in refraction at different optical zones measured with the wavefront analyzer. This is in accordance with the results of Bellucci and colleagues, who found a significant change in refraction towards myopia with spherical lenses, but not with an aspheric lens [START_REF] Bellucci | Comparison of wavefront aberrations and optical quality of eyes implanted with five different intraocular lenses[END_REF]. Denoyer et al. found a change in refraction of -0.56 D with a spherical IOL in mydriasis, while it was significantly smaller (-0.07 D) with the aspheric Tecnis ® Z9000 [START_REF] Denoyer | Quality of vision after cataract surgery after Tecnis Z9000 intraocular lens implantation; effect of contrast sensitivity and wavefront aberration improvements on the quality of daily vision[END_REF]. We found no difference when measuring best corrected visual acuity at 3 and 5 mm pupil sizes. Neither the refraction nor the visual acuity were affected at 5 mm pupil size.

Spherical aberration

The spherical aberration of the cornea was higher at 6 mm than at 3 mm optical zone. At the 6 mm optical zone the mean spherical aberration was +0.27 µm and is thus perfectly compensated by the negative spherical aberration of -0.27 µm of the Tecnis ® 1-piece. But even at the 3, 4 and 5 mm optical zones the positive corneal spherical aberration was significantly reduced by the aspheric lens (figure 2), since the spherical aberration of the lens is -0.02, -0.05 and -0.13 for the 3, 4, and 5 mm optical zones respectively (personal communication Henk Weeber, Abbott Medical Optics, Ettlingen Germany). Nevertheless, the standard deviation becomes larger with larger pupil sizes, both for the cornea and the total eye, which leads to the spherical aberration in some eyes being undercorrected or overcorrected. Thus, individual selection of the spherical aberration of the IOL based on the corneal spherical aberration may improve the results after aspheric IOL Implantation [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF][START_REF] Kohnen | Aspheric intraocular lenses[END_REF].

But even if the spherical aberration of the IOL was not individually selected in this study, the "standard" negative spherical aberration of the Tecnis ® ZCB00 fits for the majority of patients, resulting in a mean total spherical aberration of 0.0 µm.

Contrast sensitivity

In this study contrast sensitivity was not related to the pupil size in patients with an aspheric IOL (figure3). At the spatial frequency of 6 cycl/deg the contrast sensitivity did not worsen with larger pupil sizes, demonstrating that quality of vision stays as good as at small pupil sizes. Although this has not been explicitly evaluated for spherical lenses, the fact that in most studies [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF] the better contrast sensitivity of eyes with aspheric lenses is more evident under mesopic conditions may indicate a reduction of contrast sensitivity at large pupil sizes.

The study is limited by the fact that only one spatial frequency was measured. We chose this spatial frequency, because some studies could not detect a difference between aspheric IOLs at high or low spatial frequencies but mostly at a spatial frequency of 6 cycl/deg [START_REF] Montés-Micó | Analysis of the possible benefits of aspheric intraocular lenses: review of the literature[END_REF].

Defocus profiles

Theoretically, one might assume that the depth of focus is compromised by aspheric lenses.

In an eye model without any aberrations the image quality is higher at best focus than with additional spherical aberration, but the image quality deteriorates earlier with defocus [START_REF] Bühren | Application of wavefront analysis in clinical and scientific settings. From irregular astigmatism to aberrations of a higher order--Part I: Basic principles[END_REF].

But there are some important differences between a theoretical model and a clinically measured depth of focus: In a clinically measured defocus curve not only the spherical aberration, but also the other higher order aberrations have an impact on the depth of focus; also the pupil size does. Furthermore, a small pupil size may have an additional pinhole effect resulting in a higher depth of focus. In our study we could find such a pinhole effect at 3 mm pupil size, albeit faint: The depth of focus at 3 mm pupil size was only slightly broader than at 5 mm pupil size without statistically significant difference. At both pupil sizes the plateau in the defocus profiles of the Tecnis ® 1-piece resulted in a visual acuity above 20/32 at a defocus of -1 D and above 20/50 at -1.5 D (figure 4). Rocha at al. found a lower distance-corrected near visual acuity in patients with aspheric IOLs [START_REF] Rocha | Spherical aberration and depth of focus in eyes implanted with aspheric and spherical intraocular lenses; a prospective randomized study[END_REF], but they did not perform a correlation with the spherical aberration and the pupil size of the eyes [START_REF] Beiko | Spherical Aberration and Depth of Focus[END_REF][START_REF] Packer | Depth of Focus Ophthalmology[END_REF].

Since the theoretically compromised depth of focus of aspheric lenses is still under discussion, further comparisons with spherical IOLs at different simulated pupil sizes are needed.

In summary, we were able to show that after implantation of an aspheric Tecnis ® 1-piece IOL visual acuity, contrast sensitivity, depth of focus and spherical aberration are not -in contrast to spherical IOLs-compromised at large pupil sizes. This should, however, not mislead to conclude that only patients with large pupil sizes profit from an aspheric Tecnis ® 1-piece IOL.

Certainly, miotic pupil states (e.g. senile) might limit the beneficial effect of aspheric IOLs [START_REF] Awwad | Contrast sensitivity and higher order aberrations in eyes implanted with AcrySof IQ SN60WF and AcrySof SN60AT intraocular lenses[END_REF][START_REF] Kohnen | Aspheric intraocular lenses[END_REF], but one should take into account that the mean pupil size in the dark can still be 5 mm at the age of 60 and stay over 4 mm at the age of 80 [START_REF] Loewenfeld | The Pupil: Anatomy, Physiology and Clinical Applications[END_REF].

In our study the compensation of the spherical corneal aberration by the Tecnis ® 1-piece IOL was already statistically significant at a pupil size of 3 mm. Furthermore, two earlier studies could show that the total spherical aberration of the eye is significantly lower at 3 -6 mm pupil sizes in eyes with an aspheric IOL (AcrySof SN60WF-IQ and Tecnis ® Z9000) compared to a spherical IOL [16,[START_REF] Kasper | Intraindividual comparison of higher-order aberrations after implantation of aspherical and spherical intraocular lenses as a function of pupil diameter[END_REF].

In fact, three studies found a better mesopic contrast sensitivity event though the pupil sizes highly varied from 3.2 to 5.1 mm [START_REF] Awwad | Contrast sensitivity and higher order aberrations in eyes implanted with AcrySof IQ SN60WF and AcrySof SN60AT intraocular lenses[END_REF][START_REF] Denoyer | Quality of vision after cataract surgery after Tecnis Z9000 intraocular lens implantation; effect of contrast sensitivity and wavefront aberration improvements on the quality of daily vision[END_REF][START_REF] Mester | Comparison of the AcrySof IQ aspheric blue light filter and the Acrysof SA60AT intraocular lenses[END_REF][START_REF] Mester | Impact of a modified optic design on visual function: clinical comparative study[END_REF]. Mester et al found a better mesopic contrast sensitivity for the aspheric Tecnis ® Z9000 even at a mesopic pupil size of 3.2 ±0.61 mm [START_REF] Mester | Impact of a modified optic design on visual function: clinical comparative study[END_REF].

In conclusion, we were able to show that pupil size does not influence the quality of vision after implantation of an aspheric Tecnis ® 1-piece IOL. Further evaluation, however, of optical parameters with standardized, simulated pupil sizes is needed to judge the influence of pupil sizes in eyes with spherical IOLs and also to assess the benefit of aspheric IOLs in eyes with small pupils. The residual refractive error (spherical equivalent) as measured with the wavefront analyzer for 3, 4, 5 and 6 mm optical zone. There was no statistically significant difference.

Figure legends:

Figure 2:

Corneal spherical aberration (grey line) and total spherical aberration (black line) for 3, 4, 5 and 6 mm optical zones. The total spherical aberration and the corneal aberration are highly significantly different for all optical zones (P < 0.001). Thus, the negative spherical aberration of the Tecnis ® ZCB00 corrects the positive corneal aberration in all optical zones. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO

Figure 1 :

 1 Figure 1:

Figure 3 :

 3 Figure 3: Monocular contrast sensitivity under mesopic (6 cd/m 2 ) and photopic (85 cd/m 2 ) conditions

Figure 4 :

 4 Figure 4:

  editions and any other BMJPGL products to exploit all //group.bmj.com/products/journals/instructions-for-authors/licence-forms/).Competing Interest: Peter Szurman received funding by AMO, Ettlingen, Germany All other authors have no competing interests.

	subsidiary	rights,	as	set	out	in	our	licence
	(http: