CELL DENSITY REDUCTION OF bNOS IMMUNOREACTIVE AMACRINE CELLS IN MYOPIC GUINEA PIG RETINAS

José M. Romero del Hombrebueno1,2,3, Eun-Jin Lee2, Guang Zeng3, Joaquín De Juan1, Norberto M. Grzywacz2, Sally A. McFadden3

1. Dept Biotecnologia, Universidad de Alicante, Spain 2. Dept. of Biomedical Engineering, University of Southern California, Los

Angeles, USA 3. School of Psychology, University of Newcastle, Australia.

PURPOSE

Myopia is associated with the progressive andexcessive elongation of the ocular globe, andevidence suggests a retinal locus of control.Although some signaling candidates have beenproposed, the molecular mechanisms underlyingthe control of eye size in myopia are not known.Nitric oxide (NO) is a feasible candidate by itsneuromodulator role in the retina. In chicks, anitric oxide synthase (NOS) inhibitor (L-NAME)blocks form deprivation (FD) myopia1 and thereis a reduction in iNOS mRNA expression in FDeyes2. In addition, NOS activity seems to varyduring development of FD myopia in guinea pigeyes3. We report here the density anddistribution of bNOS type I and bNOS displacedamacrine cells in the retinas of myopic guinea pigeyes.

METHODS

Myopia was induced in young guinea pig eyesusing -6D lenses worn on one eye from 6 days ofage for 10 days under a 12hr/12hr light/darkcycle. Controls were performed by using eitheruntreated fellow eyes of myopic animals or 0D(plano) lenses treated eyes. At P16, refractiveerror was measured in cyclopleged eyes andocular length measured with high frequencyultrasound (Figure 1). Retinas were thenextracted and the expression pattern anddistribution of bNOS immunoreactive neuronswas studied by immunocytochemistry. Cells werecounted in images of immunoreactivity in retinalwholemounts (n=18) taken every 1mm ofeccentricity for each of 8 radial directions fromthe optic nerve head. We report here thedistribution and density of NOS Type I and NOSdisplaced amacrines cells. Data are presented asthe difference between the treated and felloweye.

Ocular measurement method

Figure 1. Schematicrepresentation of the eyemeasurement method employedin this study. At P16, after 10days of lens attachment,refractive error was measured inguinea-pig cyclopeged eyes bystreak retinoscopy, while ocularaxial length measurement wasperformed by high frecuencyultrasound.

RESULTS

Refractive error and ocular length measurements

bNOS immunoreactivity in the guinea pig retina

bNOS immunoreactive cells densitydistribution in control eyes

NOS displaced amacrine cell isodensity map in the myopic retina

Ecc

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icity f

rom

Optic

nerv

e (

mm

)

Eccentricity from Optic nerve (mm)

CONTROLMean Fellow Eye

MYOPICMean -6D Eye

NOS displaced amacrine cell difference between treated-fellow eye

Cell density distribution in myopic and plano retinas relative to untrated fellowcontrols

Figure 2. Refractive error and ocular length differences between eyes wearing a -6D or 0D (plano) lens and their respective untreated fellow eyes after 10 days oftreatment. (A) Mean refractive error differences. (B) Mean ocular lengthdifferences.

Figure 3. bNOS immunoreactivity in the guinea pig retina. Photomicrographs takenfrom 20-µm-thick vertical cryostat sections (A). The inset rectangle shows a highmagnification view of a displaced amacrine cell (small open arrow). NOSPhotomicrographs taken from whole mount retinas, at the inner margin of the INL,showing Type I NOS cells (B) and at the level of the GCL, showing displaced NOSamacrine cells (C, D). Type I NOS cell (arrows); Type II NOS cell (arrow heads);NOS displaced amacrine cell (open arrows); NOS bipolar cell (open arrow heads).OPL, outer plexiform layer; INL, inner plexiform layer; IPL, inner plexiform layer;GCL, ganglion cell layer. Scale bars 50 µm (A, B, C); 25 µm (D); 10 µm inset in (A).

Figure 5. Average Isodensity maps of NOS displaced amacrine cells between eyes wearing a -6D lens (B) and their respective untreated eyes (A) after 10 days of treatment. Myopic retinasshow a significative density reduction when compared with fellow control eyes in the ventralretina. Sample size: N=5 (5 -6D 5 fellow), each retina counted in 64 discrete regions.

Figure 6. Difference in the population of NOS displaced amacrine cells between eyes wearinga -6D lens (A) or plano lens (B) and their respective untreated eyes after 10 days oftreatment. Myopic retinas show a significative density reduction mainly in the ventral retinawhen compared with control eyes, while retinas treated with plano lenses present nodifferences with their control fellow eyes. Sample size: N=9 (5 -6D, 5 fellow; 4 plano, 4fellow), each retina counted in 64 discrete regions.

Figure 4. Average cell density of NOS Type I and displaced amacrine cells in thenon-treated guinea pig retina (n=5 wholemounts, each counted in 64 discreteregions). Both cell types significantly decline with eccentricity. Most of the NOSdisplaced amacrine cells are located in ventral and ventral-temporal retina, whichobserves the upper visual field in front of the animal. In contrast, there was nosignificant variation for different retinal sectors in the density of NOS Type Iamacrine cells.

Figure 7. Cell density differences for NOS displaced amacrine cells (A, B, C) and for NOStype I cells (D, E, F) in the dorsal, dorso-temporal, temporal, temporal-ventral, ventral,ventro-nasal and nasal retinal sectors (A, D), in the retinal eccentricity from the opticnerve (B, E), and in the whole retina taken together all eight retinal sectors (C, F).Significant differences were found between -6D and plano-treated animals for NOSdisplaced amacrine cells in the nasal, nasal-dorsal, temporal, temporal-ventral, ventral andventro-nasal retina. Additionally NOS type I cell density differences were found in thetemporal and temporal-ventral retina. *p<0.05 **p<0.01, ***p<0.001.

CONCLUSIONS

The reduction in NOS immunoreactive displacedamacrine cells in the myopic mammalian eye,suggests that bNOS is significantly down-regulated in rapidly growing eyes and implicates

these cell types in the control of myopia.

REFERENCES

1. Nickla DL, Wildsoet CF, 2004. The effect of the nonspecific nitric oxidesynthase inhibitor NG-nitro-L-arginine methyl ester on the choroidalcompensatory response to myopic defocus in chickens. Optom Vis Sci.81(2):111-8.

2. Fujii S, Honda S, Sekiya I, Yamasaki M, Yamamoto M, Saijoh K, 1998. Differentialexpression of nitric oxide synthase isoforms in form-deprived chick eyes.Curr Eye Res. 17(6):586-93.

3. Jie W, Qiong L, Xiao Y, Hui Y, Xin-mei W, Jun-wen Z, 2007. Time-course ofchanges to nitric oxide signaling pathways in form deprivation myopia inguinea pigs. Brain Res. 1186: 155-163

ACKNOWLEDGMENTSWe thank Yerina Ji and Ping Wang for technical support. We also thank Hannah Bowery, Amelia Leotta,Nadav Ivzan, Xiwu Cao and Junkwan Lee for their comments. The work was supported by James H.Zumberge Research and Fight for Sight Grants to E-JL and a University of Newcastle Grant G0900214 to

Sally Mc Fadden.

Contact information: Sally.mcfadden@newcastle.edu.au; eunjinl@bmsr.usc.edu;jossemannu@hotmail.com

A B

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N N

V VA B

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icity f

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e (

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Eccentricity from Optic nerve (mm)

-6D Difference PLANO Difference

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** ***** *** ****** ***

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A B C

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Type I

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NOS Type I Displaced NOS

Accommodation in young adults with anisometropia induced with a contact lens monovision prescription

Helen Owens & John R Phillips, Optometry & Vision Science, The University of Auckland,New Zealand

PurposeTo investigate the accommodative responses of young adults fitted with a contact lens monovision prescription.

Results: role of convergence

Methods: accommodation

Methods: Convergence

ConclusionsPre-presbyopic subjects (mean age 24.6 +/- 1.9 yrs) wearing CL monovision tended to read with the distance-corrected eye, whether the dominant or non-dominant eye was near-corrected. This was in spite of the fact that without accommodating, the target would have been in focus in the near corrected eye. However, with the lowest add used (+2.00D) and at the highest accommodative demand (4.00D) the accommodative response appeared to provide approximately equal defocus in the two eyes. The effect of reducing convergence demand with prisms, suggested that convergence played a less important role than other factors (e.g. proximity) in driving accommodation under these conditions.

Results: monovision

Acknowledgements: Karlee Foley, Andrew Gerrie, Celine Wong, Susanna Park and Trent Holden for data collection and funding from The New Zealand Optometric & Vision Research Foundation.

* Nonake F, Hasebe S, Ohtsuki H. Convergence accommodation to convergence (CA/C) ratio in patients with intermittent exotropia and decompensated exophoria. Jpn J Ophthalmol. 2004:48(3);300-5

Accommodation was measured with an open-field autorefractor (Shin-Nippon SRW-5000) in 10 young pre-presbyopic subjects (mean age: 24.6 +/- 1.9 yrs; mean Rx: -1.72 +/- 1.55 D) fitted (acutely) with a contact lens (CL) monovision prescription (+2.00D or +4.00D adds). Subjects read near letter charts at 25cm, 50cm and a Snellen chart at distance with (a) the dominant and (b) the non-dominant eye corrected for distance.

With a +4.00 D add, subjects accommodated to read targets at 25 cm or 50 cm with the distance-corrected eye, whether the dominant or non-dominant eye was corrected for near. In each case, lag of accommodation (in the distance-corrected eye) was not significantly different than with both eyes fully corrected (mean 0.36 D vs 0.32 D; P = 0.23).With a +2.00 D add, this was also the case for reading at 50 cm, but not when reading at 25cm. Then, the accommodative response resulted in defocus of approximately equal magnitude (~1.00D) but opposite sign in the two eyes (hyperopic defocus in the distance-corrected eye and myopic defocus in the near-corrected eye).

Convergence-Accommodation / Convergence (CA/C) ratios, measured using a non-accommodative target at 50 cm showed that CA/C ratios were within normal limits: 0.48 +/- 0.26 D/MA (Nonaka et al 2004*) .

To test whether convergence contributed to the accommodative response, randomly selected Base-In prisms (up to 17 ) were added in front of the near-corrected eye (+2.00 D add only) when reading at 50cm and accommodation was recorded in the distance corrected (dominant) eye. Measures were repeated with the non-dominant eye corrected for distance.

Fdist

Fnear

dist-corr

near-corrshin nippon

autoref distance

target

Fdist

Fnear

dist-corr

near-corrshin nippon

autoref 50 cm

target

Fdist

Fnear

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near-corrshin nippon

autoref 50 cm

target

-1012345678910

Focal distance (D)

ND

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D (+2)

ND

D

ND (+2)

D

ND (+4)

D

0.0

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1.0

1.5

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0 5 10 15

base-in prism (Prism dioptres)

ac

co

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od

ati

on

(D

)

rea

din

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t 5

0 c

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n=13

In most subjects, addition of base-in prism (up to 10 ) did not alter the accommodative status when reading at 50 cm with monovision with a 2.00 D add.

Commercial Relationships: First Author: None. Second Author: None

AssociAtion between PeriPherAl refrActive error Profiles for MyoPiA risk fActors

#82Judy kwan,1 Padmaja sankaridurg,1,2,3 Percy lazon de la Jara,1,2 Xiang chen,1,2,4 cathleen fedtke,1,2,3

leslie Donovan,1,2 Arthur ho,1,2,3 Jian Ge,4 brien holden,1,2,3 the vision crc Myopia control study Group

1 the brien holden vision institute, sydney, Australia; 2 the school of optometry and vision science, Unsw, sydney, Australia; 3 the vision cooperative research centre, sydney, Australia; 4 Zhongshan ophthalmic centre, Guangzhou, Peoples’ republic of china

Nasal-temporal asymmetry was consistent and did not show variation across the risk factors that were assessed, with the •exception of J45 and gender.

Field of curvature was more hyperopic for females, those with at least 1 myopic parent and for those with greater than -2.50D of •central myopia.

There was no significant differences in field curvatures of M for age which is consistent with findings by Atchison et al.• 6 indicating no differences in peripheral refraction.

Certain risk factors have been shown to be associated with a different asymmetry and/or curvature in the power vector peripheral •refractive error profile.

Further investigation in peripheral refractive errors profiles (asymmetry and curvature) will improve our knowledge of factors •associated with development and progression of myopia.

Particular risk factors (age,1 gender,1 parental myopia,2 phoria3 and degree of refractive error) have been suggested to play a role in the progression and development of myopia.

The type and profile of peripheral refractive errors have also been implicated to play a significant role in refractive error development.4

It is not known if these risk factors influence the peripheral refractive error profiles.

introDUction

To determine associations, if any, between peripheral refractive error profiles and risk factors for myopia in a group of Chinese Children

PUrPose

Centralandperipheral(nasalandtemporalvisualfieldsat20˚,30˚and40˚)cycloplegicrefractiveerrorsweremeasuredin •

545 myopic Chinese children using a modified auto-refractor (Shin Nippon NVision K5001, Japan).

Childrenwereagedbetween7-14years;refractiveerrorssphere-0.75Dto-3.50Dandcylinder≤1.00D.•

The parents’ refractive status were collected from an interview.•

Phoria was measured with a Maddox Wing (Keeler, UK), without cycloplegia, was captured using the best vision sphere. •

The investigation was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the local •

ethics committee. Informed consent was obtained from all participants and/or their guardian.

DATA ANALYSISRight eye sphero-cylindrical auto-refraction values were transformed into power vectors (M, J• 0 and J45) using the formulae of

Thibos et al.5

Powervectorperipheralrefractiveerrorprofileswereanalysedbyriskfactors;age(<11yrsvs≥11yrs),gender(femalevs•

male), parental myopia (none vs at least 1 myopic parent), phoria (eso vs exo vs ortho) and degree of central refractive error

(≤-1.50Dvs>-1.51Dto-2.50Dvs>-2.50D).

Nasal-temporal asymmetry (1• st order trend) and curvature (2nd order trend) were calculated for each risk factor and its associated

power vector peripheral refractive error profile.

Associations between the 1• st and 2nd order terms and the risk factors were assessed using linear mixed models.

Statistical significance was set to • p < 0.05.

MethoDs AnD MAteriAls

resUlts

DiscUssion AnD conclUsion

Hyman L, et al. Arch Ophthalmol 2005; 123: 977-987. 1. Mutti DO, et al. Invest Ophthalmol Vis Sci 2002; 43: 3633-36402. Goss DA, Jackson TW. Optom Vis Sci 1996; 73: 269-2783. Seidemann A, et al. J Opt Soc Am A Opt Image Sci Vis 2002; 19: 2363 23734. Thibos LN, et al. Optom Vis Sci 1997; 74: 367-3755. Atchison DA, et al. Vision Res 2005; 45: 715-7206.

references contAct DetAils

Funded by the Vision CRC through the Australian Federal Government through the CRC program, the Brien Holden Vision Institute and CIBA VISION. The authors also gratefully acknowledge the statistical support provided by Dr. Thomas Naduvilath.

J.kwan@brienholdenvision.org

AcknowleDGeMents

ASYMMETRY: 1st ORDER TREND

Nasal-temporal asymmetry was similar for all myopia risk factors associated with M and J• 0 (p>0.05).

Asymmetry was significantly different (• p < 0.001) for gender and the associated J45.

CURVATURE: 2nd ORDER TREND

• FieldofcurvatureforMwasmorehyperopicfor:

Females vs males (p < 0.001; Figure 1),

Those with at least 1 myopic parent vs none (p = 0.008; Figure 2),

Thosewithhighcentralmyopia(>-2.50D) (p < 0.01; Figure 3),

There was no significant difference in curvature of M for age (p = 0.675; Figure 4) and phoria (p = 0.853; Figure 5).

• 2nd order trend for J0wassignificantlydifferentforage(<11yrsvs≥11yrs)(p = 0.035).

resUlts continued

Figure 1: Gender and the associated M peripheral refractive error profile

Figure 2: Parental myopia and the associated M peripheral refractive error profile

Figure 3: Refractive Error and the associated M peripheral refractive error profile

Figure 5: Phoria and the associated M peripheral refractive error profile

Figure 4: Age and the associated M peripheral refractive error profile

-2.50

-2.00

-1.50

-1.00

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0.00

0.50

-40 -30 -20 -10 0 10 20 30 40

Ab

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(D

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)

Male Female

Temporal Field Nasal Field

*p < 0.001

-2.50

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-1.00

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-40 -30 -20 -10 0 10 20 30 40

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Neither One or both Parent myopes

Temporal Field Nasal Field

*p = 0.008

-3.50

-3.00

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-2.00

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-1.00

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<= -1.50D >-1.50 to -2.50D > -2.50D

Temporal Field Nasal Field

*p < 0.01

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-2.00

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<11 yrs >= 11 yrs

Temporal Field Nasal Field

p = 0.675

-2.50

-2.00

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-1.00

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0.00

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-40 -30 -20 -10 0 10 20 30 40

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(Dio

ptr

es)

Eso Exo Otho

Temporal Field Nasal Field

p = 0.853

Changes in peripheral refraction in children of different refractive errors and rates of myopic progression – 6 month results

Tsui-tsui Lee BSc (Hons) Optometry, Pauline Cho, PhD, FAAO, FBCLASchool of Optometry, The Hong Kong Polytechnic University

PurposesTo determine: relative peripheral refraction (RPR) in hyperopes (H),

emmetropes (E) and myopes (M) RPR changes over 6-month in these 3 groups of

subjects PR profiles & changes in these eyes showing different

rates of refractive changes (ΔSPH / 6M) Fast (F) - ΔSPH / 6M ≥ 0.5D more myopic Slow (S) - ΔSPH / 6M < 0.5D more myopic No-progression (N) - ΔSPH / 6M : no change or more hyperopic

in any amount

Methods RPR of 96 children (6 – 9 yo) were determined by a Shin-

Nippon Nvision 5001K auto-refractor 51 subjects have completed baseline and 6-month (6M) visits

5 measurements for each eccentricity measured: ±10˚ intervals from central fixation to 30˚ along horizontal field under cycloplegia

Results Myopes – RPR was significantly more hyperopic

compared to emmetropes and hyperopes at all eccentricities (p < 0.05, one-way ANOVA) (Figure 1)

Baseline RPR were not significantly different among different progression rate groups (p > 0.05, ANOVA)

Changes in RPR over 6-month were not significant among: the 3 refractive groups at any eccentricity the 3 progression rate groups at all eccentricities except temporal

30˚ (p > 0.05, one way ANOVA)

At ±20˚ and ±30˚ (Figure 2) Fast and slow progression groups – more hyperopic RPR No-progression group – less hyperopic RPR

No-progression group demonstrated myopic RPR within central 40˚ field

Emmetropes who (Figure 3) Developed myopia in 6M (ΔMyopia = 0.68D ) – larger increase in

hyperopic RPR Remained emmetropic in 6M (Δmyopia = 0.21D) – less increase in

hyperopic RPR

Conclusions Myopes had a hyperopic RPR profile which

was significantly different from non-myopes

Although RPR and its changes were not significantly different among groups with different progression rates, larger hyperopic RPR were observed with faster progression rate and in emmetropes who developed myopia in 6 months

PR appears to play an important role in myopic development and progression

This study was supported by the Hong Kong Polytechnic University (PolyU) PhD studentship (RGVM), a Collaborative Research Agreement between PolyU and Menicon Co, Japan and a Niche Area Funding (J-BB7P) from PolyU. We also thank Hong Kong Optical for sponsoring optical frames and lenses used in this study

Figure 1. RPR in hyperopes (H), emmetropes (E) and myopes (M)

Figure 2. RPR in fast (F), slow (S) and no (N) progression groups

Figure 3. Change in RPR in emmetropes who became myopic (E – M) and who remained emmetropic (E – E) over 6 months

Ying Yung Tang, Wing Chun Tang, Chi Ho To, Carly SY Lam

Background

The use of Shin-Nippon NVision-K has shown to be highly valid, repeatable, and

correlated with subjective refraction1. The exact mechanism behind the calculations of the

representative value (RV) adopted by the Shin-Nippon NVision-K 5001 autorefractor was

not disclosed to users. However, the RV provided by the built-in system of the

autorefractor does not equal to the manual calculated averages using individual

autorefractor outputs. Further understanding of how accurate is the RV is necessary.

Methods

The refractive error of 202 myopic eyes from 101 children aged 8 to 13 years (mean age =

10.9 years) was measured under cycloplegia using the Shin-Nippon NVision-K 5001

autorefractor. Ten autorefractor measurements were taken for both eyes of each subject.

The spherical equivalence (SE), spherical component (Sph) and cylindrical component

(Cyl) of each eye were calculated by averaging ten consecutive measurements (Mean SE,

Mean Sph and Mean Cyl) and by the vector representation method2 (Vector SE, Vector

Sph and Vector Cyl). The biases between different representation methods (the mean

difference, standard deviation, and 95% confidence intervals) were calculated as

suggested by Bland and Altman3. These calculated values were then compared with those

of representative values (RV SE, RV Sph and RV Cyl) provided by the built-in system of

the Shin-Nippon NVision-K 5001 autorefractor using one-way ANOVA with Bonferroni

adjustment for multiple comparisons.

Results

The SE value of the subjects ranged from -5.37 to -0.62D. Table 1 shows the SE, Sph, and

Cyl obtained by the three different representation methods. The Mean SE was exactly the

same as Vector SE based on the calculation formula. The RV SE from Shin-Nippon

NVision-K 5001 was not significantly different from Mean SE and Vector SE (differences =

-0.01D + 0.06; One-way ANOVA, p = 0.99). Figure 1A shows the difference in the SE

between Mean SE (or Vector SE) and RV SE compared with their mean. For all subject

eyes, the absolute difference in SE among these three representation methods was

< 0.25D (Figure1B). There were no statistically significant difference in Sph among the

three representation methods (difference between RV Sph and Mean Sph = -0.01 + 0.05;

difference between RV Sph and Vector Sph = -0.04 + 0.06) (One-way ANOVA, p = 0.92).

There were also no statistically significant differences in Cyl among the three

representation methods (difference between RV Cyl and Mean Cyl = 0.00 + 0.07;

difference between RV Cyl and Vector Cyl = 0.05 + 0.09) (One-way ANOVA, p = 0.22).

Conclusions

Although the exact mechanism behind the calculations of the representative value (RV)

adopted by the Shin-Nippon NVision-K 5001 autorefractor was not disclosed to users, we

found that it is comparable to the refraction results calculated by other conventional methods.

Therefore, the RV can serve as a convenient tool to use in clinical practice and for data

analysis in vision science research.

Acknowledgement

RGC GRF (B-Q04G) and Niche Areas Fund (J-BB7P) from The Hong Kong Polytechnic

University

References

1. Davies, L.N., Mallen, E.A., Wolffsohn, J.S. and Gilmartin, B. (2003) Clinical evaluation of

the Shin-Nippon NVision 2001/Grand Seiko WR-5001K autorefractor. Optom Vis Sci. 80,

320-324.

2. Thibos, L.N., Wheeler, W. and Horner, D. (1997) Power vectors: and application of

Fourier analysis to the description and statistical analysis of refractive error. Optom Vis

Sci. 74, 367-375.

3. Bland, J.M. and Altman, D.G.. (1986) Statistical methods for assessing agreement

between two methods of clinical measurement. Lancet. 1, 307-310.

Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University

How Representative is the Representative Value

from Shin-Nippon NVision-K 5001 Autorefractor?

Purpose

To evaluate the closeness of the representative values obtained from the Shin-Nippon

NVision-K 5001 with the refractions calculated by other conventional methods.

Table 1. Mean variability + SD (D) for each component on three representation methods.

Figure1B. Comparison of the frequency of difference between Mean SE (or Vector SE) and

RV SE (n=202 eyes).

.

Figure 1A. Difference between Mean SE (or Vector SE) and RV SE. The mean bias and 95%

confidence limit of the SE were similar.

.

0

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Averaging ten

consecutive

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representation

Spherical equivalence (SE) -2.87 + 0.98 -2.88 + 0.99 -2.88 + 0.99

Spherical component (Sph) -2.58 + 0.94 -2.59 + 0.94 -2.61 + 0.94

Cylindrical component (Cyl) -0.58 + 0.33 -0.58 + 0.32 -0.53 + 0.33