Alan P Rotchford
email: rotchford@doctors.org.uk

Rakesh Jayasawal

Savitha Madhusudhan

Anthony J King

Stephen A Vernon

Shu Fen Ho

Dr A P Rotchford Consultant Ophthalmologist

Transscleral diode laser cycloablation in patients with good vision

Keywords: Diode laser, Cyclophotocoagulation, Cyclodiode, Glaucoma

INTRODUCTION

Transscleral cyclophotocoagulation is a well-established treatment for glaucoma that has not responded to other interventions and it is widely used in advanced cases with minimal visual potential [START_REF] Vernon | Diode laser cycloablation in adult glaucoma: long-term results of a standard protocol and review of current literature[END_REF] . There is concern, however, regarding the adverse effects of this type of destructive surgery on visual function. This concern has prevented its wider acceptance as a treatment in eyes with good residual visual acuity [START_REF] Pastor | Cyclophotocoagulation: a report by the American Academy of Ophthalmology[END_REF] . However, the majority of published data relate to the treatment of eyes with initially poor vision and few series include significant numbers of cases with good visual acuity (VA). In this study we describe the visual outcome in patients undergoing transscleral cyclodiode with pre-treatment acuity of 20/60 or better.

METHODS

The study involved a retrospective case note review of all patients who underwent cycloablation with the Oculight Sx semiconductor diode 810nm laser and the contact G-probe (Iris Medical Instruments, California, USA) at Queen's Medical Centre, Nottingham, UK between April 1994 and December 2006. The authors identified patients by a review of operating theatre records in which all cases undergoing surgery are recorded. We accessed the case notes and included all patients with pre-operative best corrected Snellen visual acuity (BCVA) of 20/60 or better on at least 2 consecutive occasions at the time of treatment.

Indications for cycloablation included medically uncontrollable glaucoma in subjects who declined, had not responded to, or were unlikely, in the judgement of the surgeon responsible at the time, to respond to filtration surgery.

We performed cyclophotocoagulation under peribulbar, retrobulbar or general anaesthesia using a standard treatment protocol which we have previously described [START_REF] Spencer | Cyclodiode": results of a standard protocol[END_REF] . Briefly, this involves the application of 14 burns of 2 Watts power and 2 seconds duration, spaced half the width of the G-probe apart around 270° of the ciliary body, unless ocular co-morbidity (eg scleral thinning) dictates otherwise. Laser energy is focussed at a point corresponding to the pars plicata, 1-2 mm behind the limbus with the heel of the probe aligned at the limbus. In the contexts of congenital glaucoma, high myopia and distortion of limbal anatomy secondary to previous surgery, we identified the position of the ciliary body prior to treatment by transillumination and adjusted the probe position accordingly if necessary.

Post-operatively, we prescribed topical steroids four times daily for 2-4 weeks. We discontinued glaucoma medications during follow-up using clinical discretion based on the intraocular pressure (IOP) reduction achieved. The decision to repeat the treatment was dependent on the magnitude of the hypotensive response and the drug load necessary to achieve a satisfactory IOP.

Study parameters included demographic details, BCVA, IOP, visual field loss, anti-glaucoma medications, diagnosis, details of prior ocular surgery and duration of follow up. We documented BCVA and IOP at the pre-operative assessment, postoperatively on day 1, at 1 week, 1 month, 3 months and each subsequent clinic visit (every 3-6 months). We defined final follow-up as the last recorded clinic visit in the time period of the study.

As the primary endpoint for the study we used a reduction in BCVA by two or more lines on two successive visits. We defined successful pressure control as a final IOP ≥6 mmHg and ≤21 mmHg irrespective of the need for topical anti-glaucoma medication but without the need for oral acetazolamide or further glaucoma surgery. We performed data analysis using SPSS V10 (SPSS Inc, Chicago, Il). We used the chi squared test for determining the statistical significance of differences between discrete variables (or Fisher's exact test when a cell value less than 5 was expected), Student's t test for parametric continuous data and Mann-Whitney for non-parametric continuous. We performed Kaplan-Meier survival analysis to investigate the loss of visual acuity following treatment.

RESULTS

Out of 351 patients recorded as undergoing cyclodiode treatment, we identified 51 eyes of 45 patients with initial visual acuity of 20/60 or better.

Two patients were lost to follow-up within the first 12 months (one died and one after discharge back to the referring centre). For the remaining 49 eyes of 43 patients, the distribution of glaucoma types and other baseline characteristics are shown in table 1. Median pre treatment BCVA was 20/30 (figure 1).

Reliable visual fields prior to cyclodiode were available for 36/49 eyes (73.5%). Of these, 25 were automated Humphrey threshold fields, 8 were Goldmann and 3 were Friedmann fields. Mean deviation (SD) for the Humphrey fields was 13.8 (9.7) dB (range 1.6 to 30.4). Advanced loss was present in 19/36 (52.7%) of fields.

We performed a total of 85 treatment episodes on these 49 eyes using a power setting of 2 Watts for a duration of 2 seconds for all shots. We applied a mean of 14.4 shots with a mean energy of 57.6 Joules per treatment episode We performed a mean of 1.73 treatment episodes per eye (range 1-6; median 1.0) with more than one episode given in 18/49 eyes (36.7%). During the course of treatment we applied a total mean energy of 99.7 Joules per eye (range 40-336; median 60J). .After the first treatment we followed subjects for a mean of 263 weeks (range 62-578), or 5.1 years.

IOP and glaucoma medication use at final follow-up is shown in table 2. IOP

was controlled at final follow-up without oral acetazolamide in 39/49 (79.6%).

None of the 49 eyes developed sustained hypotony (IOP<6 mmHg) during follow-up although one eye developed transient hypotony after both treatment episodes which resolved by the end of the first post-operative month.

Median VA at final follow-up was 20/60 (figure 1). It improved in 8/49 eyes (16.3%), remained unchanged in 11/49 (22.5%) and deteriorated by 1 line or more in 30/49 (61.2%). Final visual acuity, after a mean of 5.0 years followup, was reduced to less than 20/200 in 8/49 eyes (16.3%).

A loss of 2 Snellen lines or more of acuity was measured in15/49 eyes (30.6%) in 15 different patients. The mean (SD) survival time using this primary endpoint was 399.7 (70.3) weeks (95%CI: 330.8-468.6) or 7.7 years (figure 2). During the first 12 months 18.3% (9/49) of eyes lost 2 or more lines of acuity.

There was no difference in the mean number of treatment episodes between those losing visual acuity (1.60) and those maintaining visual acuity (1.79) (p=0.69). Loss of visual acuity was also independent of the total energy applied (p=0.66), age (p=0.11), sex (p=0.14), pre-operative IOP (p=0.63), preoperative VA (p=0.17) or advanced pre-operative visual field loss (p=0.22).

Those that lost visual acuity had a similar final IOP (15.0 mmHg) to the remainder (16.1 mmHg) (p=0.55).

In 8 cases loss of visual acuity was solely attributed to glaucoma progression and it was a contributory factor in one further case (table 2). Of these 9 cases loss of acuity occurred in spite of IOP consistently below 21 mmHg during follow-up in 4 cases, with a mean final IOP of 14 mmHg. All had advanced disc cupping and/or visual field loss affecting the central 10 degrees. In the other 5 eyes IOP was not consistently controlled following cyclodiode treatment. In one case rapid loss of visual acuity from 20/60 to 20/600 occurred 6 days after treatment. This was attributed to optic nerve "snuff out" caused by an IOP of 50mmHg.

Visual loss was attributed partly or entirely to macula oedema in 4 cases.

One had a history of proliferative diabetic retinopathy, one was aphakic and one had a history of previous vitreous loss during cataract surgery. These 3 eyes underwent a single standard treatment session of 56 Joules and the reduction in visual acuity was delayed between 6 and 12 months post treatment. In the fourth case the macula oedema developed a week after the second treatment episode.

One case was due to a rhegmatogenous retinal detachment (RD) which developed 10 weeks after cyclodiode in an eye with uveitic glaucoma. This patient had already had a spontaneous RD in the fellow eye.

One case with no identified risk factors developed a vitreous haemorrhage 5 days after cyclodiode in an eye with primary open-angle glaucoma (POAG).

By the time that this had cleared spontaneously a dense cataract had formed reducing acuity to counting fingers. The patient declined cataract surgery.

DISCUSSION

Cycloablation is a recognised therapeutic approach in the management of complex and refractory glaucoma and appears to be an effective method of achieving a sustained reduction in IOP [START_REF] Vernon | Diode laser cycloablation in adult glaucoma: long-term results of a standard protocol and review of current literature[END_REF] . Advantages over the main alternatives -filtration or drainage device surgery -include technical ease of the procedure, quicker recovery and none of the risks specific to intraocular surgery. However, cyclodestructive procedures have been linked to subsequent loss of visual acuity in a substantial proportion of those treated [START_REF] Youn | Factors associated with visual acuity loss after noncontact transscleral Nd:YAG cyclophotocoagulation[END_REF][START_REF] Kosoko | Long-term outcome of initial ciliary ablation with contact diode laser transscleral cyclophotocoagulation for severe glaucoma. The Diode Laser Ciliary Ablation Study Group[END_REF] .

As a result, this approach has generally been reserved for patients who have previously undergone multiple glaucoma operations, are poor surgical candidates or who already have limited visual potential [START_REF] Pastor | Cyclophotocoagulation: a report by the American Academy of Ophthalmology[END_REF][START_REF]Primary open-angle glaucoma, Preferred Practice Pattern[END_REF] .

Resistance to the wider application of laser cyclotherapy is based for the most part on series of cases with visual acuity significantly compromised by the glaucoma itself or, in the case of secondary glaucomas, its underlying cause such as retinal vascular disease or uveitis. Published data on the long-term prognosis for vision following cyclodiode treatment applied to patients with significant baseline visual acuity are few (table 3) [START_REF] Schuman | Contact transscleral Nd:YAG laser cyclophotocoagulation[END_REF][START_REF] Bloom | Cyclodiode". Trans-scleral diode laser cyclophotocoagulation in the treatment of advanced refractory glaucoma[END_REF][START_REF] Egbert | Diode laser transscleral cyclophotocoagulation as a primary surgical treatment for primary open-angle glaucoma[END_REF][START_REF] Wilensky | Long-term visual outcome of transscleral laser cyclotherapy in eyes with ambulatory vision[END_REF][START_REF] Ansari | Long-term efficacy and visual acuity following transscleral diode laser photocoagulation in cases of refractory and non-refractory glaucoma[END_REF] .

In this cohort of patients, the largest we have identified to date with visual acuity initially 20/60 or better, acuity was found to have declined from a median of 20/30 to 20/60. A two or more line reduction in Snellen acuity was seen in 30.6% of eyes and was reduced to less than 20/200 in 16.3%. The most frequent cause of visual loss was further progression of glaucoma which was an attributable cause in half the cases. In a number of these, this occurred despite a significant and sustained response to treatment. The only other cause linked to more than one case of visual loss was cystoid macula oedema which was a contributory factor in 3. Notably in 2 of these cases, neither of whom had an intact posterior capsule, cystoid did not develop until 6 months or more following treatment. In a further case it could be argued that cyclodiode therapy might have led to worsening of diabetic macula oedema.

In no case did persistent hypotony develop, a finding which is consistent with our previous reported experience using the relatively conservative, standardised treatment protocol employed in these patients. Macular pucker, which has previously been reported as a cause of visual loss following laser cyclotherapy [START_REF] Bloom | Cyclodiode". Trans-scleral diode laser cyclophotocoagulation in the treatment of advanced refractory glaucoma[END_REF] , was not seen clinically, although neither OCT nor fluorescein angiography were used.

Clearly it is difficult to attribute the deterioration in vision in the patients in this cohort in part or wholly to the cyclodiode treatment. Some loss may have been coincidental reflecting the natural course of the underlying disease and some might have occurred whatever surgical intervention had been applied. Nevertheless loss of 2 lines of visual acuity in 30% over a mean 5 year followup period is concerning. However, other treatment options also carry a significant risk to vision. In the recent tube versus trabeculectomy (TVT) study, patients with medically uncontrolled glaucoma who had previously undergone cataract and/or filtration surgery were randomised to insertion of a glaucoma drainage device or augmented trabeculectomy [START_REF] Gedde | Treatment outcomes in the tube versus trabeculectomy study after one year of follow-up[END_REF] . Mean baseline visual acuity was equivalent to that in our cohort before cyclodiode. A 2 or more Snellen line reduction in acuity was observed in 32% in the tube arm and 33% in the trabeculectomy group after 12 months. Even after excluding cataract as a cause the figures were 27% and 24% respectively. The equivalent one year figure for our cohort was 18% despite inclusion of a large number of patients with chronic uveitis, aphakia and other secondary glaucomas which were excluded from the TVT study. Similarly, in the Advanced Glaucoma Intervention Study, 40% of medically uncontrolled patents treated with either trabeculectomy or laser trabeculoplasty had a 3line reduction in visual acuity during 5 years follow-up [START_REF]The Advanced Glaucoma Intervention Study (AGIS): 4. Comparison of treatment outcomes within race. Seven-year results[END_REF] . Loss of visual acuity is, therefore, by no means the preserve of any single surgical modality.

The earlier use of laser cyclotherapy in eyes with good residual visual acuity appears to be gaining support [START_REF] Wilensky | Long-term visual outcome of transscleral laser cyclotherapy in eyes with ambulatory vision[END_REF][START_REF] Ansari | Long-term efficacy and visual acuity following transscleral diode laser photocoagulation in cases of refractory and non-refractory glaucoma[END_REF] . Ansari et al presented a retrospective subgroup of 23 eyes with POAG and mean pre treatment visual acuity in the range 20/20 to 20/120) who underwent a single application of transscleral cyclodiode between 60 and 160 Joules (mean 120J) [START_REF] Ansari | Long-term efficacy and visual acuity following transscleral diode laser photocoagulation in cases of refractory and non-refractory glaucoma[END_REF] . Mean visual acuity did not deteriorate although the final visual acuity was worse in 3 cases (13%), 2 of which were due to cataract progression.

In another retrospective study of 21 eyes with pre-laser visual acuity 20/80 or better which had previously mostly undergone filtration surgery, cycloablation was applied either by diode or Nd:YAG laser [START_REF] Wilensky | Long-term visual outcome of transscleral laser cyclotherapy in eyes with ambulatory vision[END_REF] . Although 17 of the 21 patients required additional laser and/or surgical treatment to further lower IOP, visual acuity remained within one line of the pre-treatment level in 81%.

A prospective study in Ghana even examined the effect of cyclodiode as a primary surgical treatment in one eye of patients with POAG [START_REF] Egbert | Diode laser transscleral cyclophotocoagulation as a primary surgical treatment for primary open-angle glaucoma[END_REF] . Although a reduction in visual acuity was observed in 18 (23%) after a mean follow-up of 13.2 months, there was no significant difference in acuity between the fellow eyes treated medically and the diode treated eye. In the 19 eyes with pretreatment visual acuity 20/60 or better, only 1 had a decrease in visual acuity.

Our study's main limitations are its retrospective nature and the absence of a control group to distinguish the adverse effects of the treatment from the natural history of the underlying disease. While undertaking cyclodiode treatment in eyes with uncontrolled glaucoma but with good visual acuity comes with a significant risk of visual loss, that risk does not appear to be any greater than for other, more conventional surgical interventions. This needs testing in a randomised study.

In the absence of such comparative data, this study indicates that cyclodiode treatment can be used effectively in eyes with good visual acuity and may be a useful alternative surgical treatment in patients with difficult to manage glaucoma.

  Visual field testing was performed by Humphrey threshold standard automated perimetry, Goldmann or Friedmann perimetry. For the purposes of this group of patients we classified visual field loss as advanced in the presence any of the following: a) Humphrey 24-2 mean deviation ≥ 15dB; b) deviation of ≥15dB affecting any part of the central 10 degrees on Humphrey 24-2; c) encroachment of the I4e isopter in the central 10 degrees of a Goldmann field; or d) any loss of the central 4 target points on Friedmann field.

Figure 1 .

 1 Figure 1. Snellen visual acuity distribution pre cyclodiode and at final follow-

Figure 2 .

 2 Figure 2. Kaplan-Meier plot showing survival time to loss of 2 or more lines of

Table 1 .

 1 Baseline characteristics

	Category	Cases (n=43)

d Includes 1 eye with aqueous misdirection. e 2 secondary to anterior chamber lens implant; 2 with pseudoexfoliation, 1 each following retinal detachment surgery, PK, trauma, rubeosis iridis.

Table 2 .

 2 IOP and glaucoma medication use at final follow-up after cyclodiode

	Outcome

Table 3 .

 3 Details of cases losing 2 or more lines of visual acuity following cyclodiode treatment.

		VA drop	Glaucoma	Total dose:	Details of loss of visual acuity
			type	shots/Joules
	1	20/60-20/100	POAG	15/60	Glaucoma progression despite IOP control
	2	20/16-20/40	POAG	14/56	Glaucoma progression despite IOP control
	3	20/60-20/200 RD surgery	14/56	Glaucoma progression despite IOP control
	4	20/60-HM	POAG	20/80	Glaucoma progression despite IOP control
	5	20/60-LP	PXF	56/224	Glaucoma progression -poor IOP control
	6	20/30-NLP	POAG	56/224	Glaucoma progression -poor IOP control
	7	20/60-NLP	Rubeotic	70/280	Glaucoma progression -poor IOP control
	8	20/60-HM	PK	26/104	ON infarct after 6 days -poor IOP control
	9	20/60-20/400	AM	31/124	CMO 1 month after 2 nd dose + glaucoma
					progression with poor IOP control
	10	20/30-20/60	Aphakic	14/56	CMO after 6 months
	11	20/30-CF	AC IOL	14/56	CMO after 12 months. Later corneal oedema.
	12	20/30-20/80	POAG	14/56	Diabetic maculopathy after 12 months
	13	20/60-CF	POAG	15/60	Cataract -declined surgery
	14	20/20-CF	Uveitic	28/112	RD 10 weeks after 2 nd dose
	15	20/30-20/80	POAG	14/56	Dry AMD
	VA (visual acuity); CF (counting fingers); HM (hand movement perception); LP (light perception); NLP
	(no light perception); POAG (primary open-angle glaucoma); PXF (pseudoexfoliative glaucoma); RD
	(retinal detachment); IOP (intraocular pressure); AM (aqueous misdirection); CMO (cystoid macula

oedema); AMD (age-related macula degeneration).

Author

No


MD Hemma Resch

MD Gabor Deak

MD Clemens Vass
email: clemens.vass@meduniwien.ac.at

Keywords: optic disc size, scanning laser polarimetry, retinal nerve fiber layer

Background: To date there is no knowledge about the impact of optic disc size on the retinal nerve fiber layer (RNFL) as measured with GDx VCC or GDx ECC. Since the size of the optic disc is not taken into account for the analyses carried out by the GDx it might affect sensitivity in large optic discs and specificity in small ones.

Methods: Topographic optic disc measurements and RNFL thickness values of 80 healthy subjects were measured using HRT3 and GDx VCC and ECC. Subjects were divided into 3 equally large groups depending on optic disc area. We used ANOVA to test the differencs between groups of the GDx VCC and ECC parameters TSNIT average, superior average, inferior average and NFI.

Results:

The mean optic disc sizes of the three groups were 1.61, 2.00, and 2.61 µm 2 . The optic disc area significantly affected none of the tested RNFL parameters of the GDx and HRT3. For all HRT3 parameters of optic disc morphology, ANOVA showed statistical significance between the three groups of different optic disc areas.

Conclusion:

We could not find a significant impact of optic disc size on any RNFL parameter measured with GDx VCC or ECC and with HRT3.

Introduction

The objective and quantitative measurement of peripapillary retinal nerve fiber layer (RNFL) is possible with scanning laser polarimetry (SLP; GDx Nerve Fiber Analyzer; Carl Zeiss Meditec, Dublin, CA) which relies on the assumption that the parallel arrangement of microtubules within the RNFL causes a change in the state of polarisation of an illuminating laser beam, known as retardation. [START_REF] Chi | Racial differences in optic nerve head parameters[END_REF][START_REF] Tjon-Fo-Sang | The sensitivity and specificity of nerve fibre layer measurements in glaucoma as determined with scanning laser polarimetry[END_REF] The amount of retardation of the incident laser light is proportional to the thickness of the tissue.

Although retardation is considered to be proportional to the thickness of the RNFL it can be affected by other polarizing ocular structures, such as the cornea, which interferes with accurate assessment of RNFL thickness. [START_REF] Weinreb | Histopathologic validation of Fourierellipsometry measurements of retinal nerve fiber layer thickness[END_REF] Accordingly, GDx with variable corneal compensation (GDx VCC) was developed for better compensation of corneal birefringence, which has better diagnostic ability than GDx with fixed corneal compensation (FCC). [START_REF] Zhou | Individualized compensation of anterior segment birefringence during scanning laser polarimetry[END_REF] Nevertheless, there is still some interference from the subretinal structures, and the image produced by GDx VCC sometimes shows an atypical retardation pattern (ARP). ARP is seen as alternating peripapillary circumferential bands of low and high retardation [START_REF] Da Pozzo | Atypical pattern of retardation on GDx-VCC and its effect on retinal nerve fibre layer evaluation in glaucomatous eyes[END_REF] and is thought to be common in eyes with a low signal-to-noise ratio resulting from thinning of the retinal pigment epithelium, such as in aged or myopic eyes. [START_REF] Bozkurt | Effect of peripapillary chorioretinal atrophy on GDx parameters in patients with degenerative myopia[END_REF] Enhanced corneal compensation (ECC) is based on the compensation method. This scheme has been developed to improve the signal-to-noise ratio and to eliminate the ARP associated artefacts. [START_REF] Toth | Enhanced corneal compensation for scanning laser polarimetry on eyes with atypical polarisation pattern[END_REF] Several studies have demonstrated the benefits of GDx ECC for the assessment of the RNFL. [START_REF] Toth | Enhanced corneal compensation for scanning laser polarimetry on eyes with atypical polarisation pattern[END_REF][START_REF] Bowd | Retinal nerve fiber layer thickness and visual sensitivity using scanning laser polarimetry with variable and enhanced corneal compensation[END_REF] Since a couple of years it is possible to have reliable sophisticated three dimensional analysis of the optic nerve head with the development of confocal scanning laser tomography. [START_REF] Chauhan | Test-retest variability of topographic measurements with confocal scanning laser tomography in patients with glaucoma and control subjects[END_REF][START_REF] Beausnecourt | Quantitative analysis of macular holes with scanning laser tomography[END_REF] In the present study we used HRT3 for quantifying the morphological optic disc parameters.

Considerable variability in RNFL thickness values among normal subjects, and some overlap in thickness values between normal subjects and glaucoma patients have been reported in several studies conducted by SLP. [START_REF] Choplin | Differentiating patients with glaucoma from glaucoma suspects and normal subjects by nerve fibre layer assessment with scanning laser polarimetry[END_REF][START_REF] Poinoosawmy | Variation of nerve fibre layer thickness measurements with age and ethnicity by scanning laser polarimetry[END_REF] The number of nerve fibers and the configuration of the optic nerve head vary considerably in the normal population. The size of the scleral canal, the tilt of the optic nerve head, and the number of the nerve fibers and their arrangement all contribute to this variation. [START_REF] Quigley | The size and shape of the optic disc in normal human eyes[END_REF] Previous studies using SLP with fixed corneal compensation (GDx FCC) found out that the peripapillary RNFL thickness increases linearly as the area of the optic nerve head increases. [START_REF] Funaki | Relation between size of optic disc and thickness of retinal nerve fibre layer in normal subjects[END_REF][START_REF] Toprak | Relation of optic disc topography and age to thickness of retinal nerve fibre layer as measured using scanning laser polarimetry, in normal subjects[END_REF] Medeiros et al. found that the diagnostic performances of the GDx VCC, HRT2, and Stratus OCT in glaucoma patients were significantly influenced by the optic disc size. [START_REF] Medeiros | Influence of disease severity and optic disc size on the diagnostic performance of imaging instruments in glaucoma[END_REF] Larger optic discs were associated with decreased sensitivity for the Stratus OCT parameter average thickness and the GDx VCC parameter nerve fiber indicator, whereas small optic discs were associated with increased sensitivity. For the Moorfields regression analysis classification measured by the HRT2, an inverse effect was observed. Also RNFL thickness measurements obtained by Stratus OCT increased significantly with an increase in optic disc size. [START_REF] Savini | Correlation between retinal nerve fibre layer thickness and optic nerve head size: an optical coherence tomography study[END_REF] To date there is no knowledge about the impact of optic disc size on the RNFL as measured with GDx VCC or GDx ECC. Since the size of the optic disc is not taken into account for the analyses carried out by the GDx it might affect sensitivity in large optic discs and specificity in small ones.

It was the aim of the present study to quantify the influence of optic disc size on the peripapillary RNFL thickness as measured with the current and upcoming SLP technologies in healthy subjects.

Subjects and Methods

Subjects

The study protocol was approved by the Ethics Committee of the Medical University of Vienna and followed the guidelines of Good Clinical Practice and the Declaration of Helsinki. 83 healthy, caucasian subjects of both sexes aged between 18 to 69 years were included.

Inclusion and exclusion criteria

Inclusion criteria were normal findings in medical history and normal ophthalmic findings, especially normal appearance of the optic disc and normal visual fields. An abnormal visual field was defined as a glaucoma hemifield test outside normal limits and/or a corrected pattern standard deviation with P<0.05. [START_REF] Keltner | Classification of visual field abnormalities in the ocular hypertension treatment study[END_REF] Any of the following excluded a subject from participation in the trial: Evidence of any eye disease except refractive error, history of ocular trauma or intraocular surgery within the last 6 months, ocular inflammation or infection within the last 3 months, pregnancy, astigmatism more than +2.0 diopters and ametropia of more than +/-5.0 diopters. Subject with tilted disc and peripapillary atrophy that extended into the measurement ring were excluded.

Experimental Paradigm

Initially a prestudy screening was carried out, where the medical and ocular history was taken. A complete ophthalmological examination was performed, including fundoscopy, visual acuity and the measurement of intraocular pressure (IOP) by Goldmann applanation tonometry. Standard automated perimetry (SAP) was performed with the Humphrey field analyser II (HFA II, program 30-2, SITA Standard, Carl Zeiss).

Subjects eligible for participating in the study according to the inclusion/exclusion criteria were included. One eye was randomly selected for the measurements.

The study was performed at the Department of Ophthalmology, Medical University of Vienna, Austria.

Methods

Automated visual field testing was performed with the Humphrey field analyzer II (program 30-2). Visual field eligibility criteria were less than 33% false-positive responses, less than 33% false-negative responses, and less than 33% fixation losses. Optic disc morphology was measured with the confocal scanning laser tomograph HRT3. Two series of 3 images each were acquired and the series with the smaller standard deviation was chosen for analysis.

Scanning laser polarimetry GDx VCC was performed using a commercial GDx system Version 5.5.0 (NDB Version 1.05.00, Carl Zeiss Meditec, Dublin, CA, USA). Additionally we used the Gdx ECC method, which was run on the same commercial GDx system specified above. The general principles of GDx have already been described elsewhere. [START_REF] Zhou | Individualized compensation of anterior segment birefringence during scanning laser polarimetry[END_REF][START_REF] Weinreb | Glaucoma detection using scanning laser polarimetry with variable corneal polarization compensation[END_REF] Briefly, SLP assesses the RNFL thickness in the peripapillary retina by measuring the RNFL birefringence with a near-infrared diode laser. With GDx ECC software, the corneal polarization compensator is automatically adjusted so that the combined retardation magnitude from the cornea and the compensating retarder is approximately 55 nm with a vertical slow axis of polarization. This adjustment bias serves to boost the signal to overcome low sensitivity that can make retardation measurements susceptible to optical and electronic noise. After image acquisition, the bias is subtracted to yield the RNFL retardation values.

In our study, each subject with pupils undilated had scans on the same day performed by an experienced operator. The spherical equivalent refractive error was tested subjectively and entered into the software to allow the GDx to focus on the retina. To optimize image quality the focus was adjusted manually in 0.25-diopter steps if necessary. All selected images were of high quality (quality scan score of 8 or greater), with a centred optic disc, were well focused and illuminated throughout the image, and were without any motion artefacts. A fixed concentric measurement band with 27 pixels (approximately 2.4 mm) inner and 35 pixels (approximately 3.2 mm) outer diameter was centred on the optic disc, after which the measurements of peripapillary retardation were conducted. Retardation was converted to an estimate of RNFL thickness by the software.

The GDx VCC and ECC parameters investigated in this study were the RNFL thickness values including temporal-superior-nasal-inferior-temporal (TSNIT) average, superior average, inferior average, ellipse standard deviation (TSNIT SD), as well as nerve fiber indicator (NFI) and typical scan score (TSS). [START_REF] Toth | Enhanced corneal compensation for scanning laser polarimetry on eyes with atypical polarisation pattern[END_REF]

Statistical methods

Subjects were divided into 3 equally large groups depending on the optic disc area as measured with HRT3 (group 1: <1.85 mm2; group 2: 1.85 mm2 to 2.23 mm2; and group 3: >2.23 mm2). Parameters of HRT, GDx VCC and GDx ECC were compared between groups by ANOVA. All statistical analysis was done with the SPSS® software package (SPSS Inc., USA) release No. 16.0.2.

Results

44 males and 36 females were included into the study. Subjects' baseline characteristics, IOP and visual field mean deviation (MD) are given in table 1. We were only able to obtain a sufficient measurement quality of 80 out of 83 subjects. Hence all data on optic nerve head morphology are from 80 subjects.

Average optic disc area measured with HRT3 was within the expected range, and as expected, with a huge variability between subjects. The mean optic disc area was 2.07 mm 2 (SD ± 0.48) and ranged from 1.17 mm 2 to 3.62 mm 2 . Table 2 shows means and standard deviations of optic disc topographic parameters as measured with HRT3 and for comparison the ophthalmoscopically determined C/D ratios (examined with the Volk Superfield lens). For all parameters of optic disc morphology, ANOVA showed statistical significance between the three groups of different optic disc areas. In contrast the HRT3 measures of the RNFL did not reach the level of significance in ANOVA.

In table 3 the parameters of GDx VCC and ECC are shown and compared between the groups of different optic disc areas. None of the tested variables depended significantly on the optic disc area.

Discussion

Reference values for retinal RNFL thickness of different ages, ethnic groups, and refractive stages are needed to define normality of RNFL thickness. Several studies have been performed with SLP on variation of RNFL thickness measurements with age and ethnicity [START_REF] Chi | Racial differences in optic nerve head parameters[END_REF][START_REF] Poinoosawmy | Variation of nerve fibre layer thickness measurements with age and ethnicity by scanning laser polarimetry[END_REF] but the effect of axial length and optic disc size on RNFL thickness measurements is still not clear.

Our data showed a tendency towards an increase of RNFL thickness measurements with an increase in optic disc size but we could not find a significant impact of optic disc size on any retinal nerve fiber parameter measured with GDx VCC or ECC and with HRT3. While this possibly might be attributed to our sample size, the power was sufficient to detect a clinically meaningful difference between groups of 10%. Contrary to our results, previous studies using Gdx FCC, HRT II, and Stratus OCT showed a significant increase of RNFL thickness by the optic disc size. [START_REF] Toprak | Relation of optic disc topography and age to thickness of retinal nerve fibre layer as measured using scanning laser polarimetry, in normal subjects[END_REF][START_REF] Medeiros | Influence of disease severity and optic disc size on the diagnostic performance of imaging instruments in glaucoma[END_REF][START_REF] Keltner | Classification of visual field abnormalities in the ocular hypertension treatment study[END_REF][START_REF] Nagai-Kusuhara | Association of retinal nerve fibre layer thickness measured by confocal scanning laser ophthalmoscopy and optical coherence tomography with disc size and axial length[END_REF] It might be that eyes with large ONHs show a thicker RNFL as a result of an increased amount of nerve fibers. [START_REF] Quigley | Larger optic nerve heads have more nerve fibers in normal monkey eyes[END_REF] Medeiros et al. have demonstrated reduced sensitivity of GDx VCC and Stratus OCT in eyes with large optic discs and have discussed that this might be an effect of increased number of optic nerve fibers in those eyes. [START_REF] Savini | Correlation between retinal nerve fibre layer thickness and optic nerve head size: an optical coherence tomography study[END_REF] Nagai et al. found a significant positive correlation of RNFL and disc size both measured with Stratus OCT, but not with disc size as measured with HRT. [START_REF] Nagai-Kusuhara | Association of retinal nerve fibre layer thickness measured by confocal scanning laser ophthalmoscopy and optical coherence tomography with disc size and axial length[END_REF] The possibly most important difference between these two instruments in determination of the disc size is that the Stratus OCT does not correct for ocular magnification error caused by refractive errors (even if they are corrected for by optical means, for example by focussing) while the HRT at least partially does. As a consequence in the Stratus, and also the GDx VCC the apparently fixed diameter of the measurement ring becomes dependent on refractive error. With OCTs and also the GDx VCC the optic discs of hyperopic individuals appear to be larger and the measurement rings unchanged, while on the retina the measurement ring is reduced in size and the optic disc may be just average. The opposite holds true for myopic subjects. While this effect may account for part of the correlation between disc size and RNFL thickness, the same holds true for our study where we did not find a correlation.

There is more than one possible interaction between apparent optic disc size in retinal imaging and refractive error. Due to different magnification the effective imaging area in myopic eyes gets larger resulting in apparently smaller disc size. Taking into account the range of refractive errors in the population studied, this may have resulted in errors of the image sizes of -7% to +7% without mathematical correction as in GDx, or -4% to +3% with the built-in correction method of the HRT. [START_REF] Garway-Heath | Measurement of optic disc size: equivalence of methods to correct for ocular magnification[END_REF] Secondly there is a correlation between axial length and the size of the optic disc, although it may be valid only for refractive errors outside -8 to +4 diopters. [START_REF] Jonas | Optic disk size correlated with refractive error[END_REF] The technical aspects of the different measurement methods might explain the controversial study results. SLP determines RNFL thickness in the peripapillary region by measuring the total retardation in the light passing through the microtubules within the ganglion cell axons. This means that it does not directly measure the thickness of the RNFL but an optical property linked to the viability of the RNFL. OCT on the other hand measures the thickness of RNFL on cross sectional images of the retina generated by low coherence interferometry. Also when comparing our results to studies using the GDx FCC [START_REF] Toprak | Relation of optic disc topography and age to thickness of retinal nerve fibre layer as measured using scanning laser polarimetry, in normal subjects[END_REF][START_REF] Medeiros | Influence of disease severity and optic disc size on the diagnostic performance of imaging instruments in glaucoma[END_REF] some differences in methods should be noted. The GDx FCC used a measurement circle of variable diameter depending on disc diameter. Thus, the circumference of this ring increases together with disc size. While the spreading nerve fibers should have similar cross-sectional area at different radius of the circle, some artefacts such as effects of corneal retardation due to the fixed cornea compensation, or atypical pattern may add to the RNFL. This addition might increase the integral of RNFL with increasing size of the measuring ring.

At least for the small optic discs another factor might also influence results of the GDx FCC.

It has been reported that the RNFL determined with GDx FCC increases from disc margin over 0.2 to 0.4 mm before reaching its maximum. In small discs the measurement ring might sometimes be too close to the disc and therefore miss the maximum RNFL thickness. However this finding has not been confirmed by others. [START_REF] Iester | Normal retinal nerve fiber layer thickness in the peripapillary region measured by scanning laser polarimetry[END_REF] As shown in a previous study with ECC [START_REF] Toth | Enhanced corneal compensation for scanning laser polarimetry on eyes with atypical polarisation pattern[END_REF], TSS was higher than with VCC, suggesting that atypical retardation patterns were less pronounced in images obtained with ECC. The origin of atypical scans is not yet fully understood. However, it appears to be associated with a low signal-to-noise ratio. In areas of low signal the measurements are influenced by electronic

Table 1

 1 Subjects baseline characteristics, IOP and visual field MD *

	3 groups depending on	Small (group 1:	Medium (group 2:	Large (group 3:
	the optic disc area	<1.85 mm 2 )	1.85 mm 2 to 2.23	>2.23 mm 2 )
			mm 2 )
	Age (years)	25.0 (±4.8)	28.6 (±10.9)	26.6 (±7.8)
	Refractive error (diopters) -0.06 (±2.03)	0.34 (±1.51)	-0.42 (±1.46)
	IOP (mmHg)	14.8 (±2.2)	14.4 (±2.3)	15.2 (±2.8)
	MD (db)	-0.18 (±0.81)	-0.17 (±0.1)	-0.04 (±0.87)
	IOP = intraocular pressure, MD = mean deviation, dB = decibel

Table 2

 2 Optic disc parameters and RNFL thickness as measured with HRT3 *

	3 groups	Small (group	Medium (group	Large	ANOVA
	depending on	1: <1.85 mm 2 )	2: 1.85 mm 2 to	(group 3:	(p values)
	the optic disc		2.23 mm 2 )	>2.23 mm 2 )
	area
	Optic Disc Area	1.61 (±0.19)	2.00 (±0.11)	2.61 (±0.37)	<0.001
	(mm 2 )
	Rim Area (mm 2 )	1.38 (±0.22)	1.71 (±1.19)	2.00 (±0.41)	<0.001
	Rim Volume	0.40 (±0.11)	0.48 (±0.13)	0.54 (±0.23)	0.008
	(mm 3 )
	C/D Area Ratio	0.14 (±0.09)	0.14 (±0.09)	0.23 (±0.12)	0.002
	Linear C/D	0.35 (±0.14)	0.34 (±0.18)	0.45 (±0.16)	0.019
	Ratio (HRT)
	Mean RNFL	0.28 (±0.06)	0.27 (±0.06)	0.25 (±0.04)	0.180
	thickness (mm)
	RNFL	1.24 (±0.28)	1.36 (±0.30)	1.42 (±0.25)	0.053
	Crossectional
	Area (mm 2 )
	C/D Ratio (Volk)	0.14 (±0.09)	0.21 (±0.12)	0.26 (±0.15)	0.003

C/D = cup/disc ratio, RNFL = retinal nerve fiber layer

Table 3

 3 Comparison of GDx parameters between groups of different optic disc area (ANOVA) * Results are presented as means ± SD (n=80). VCC = variable corneal compensation, ECC = enhanced corneal compensation, NFI = nerve fiber indicator, TSNIT = temporal, superior, nasal, inferior, temporal average, TSNIT SD = ellipse standard deviation, TSS = typical scan score

		3 groups	Small (group	Medium (group	Large (group	ANOVA
		depending on the	1: <1.85m	2: 1.85 mm 2 to	3: >2.23	(p values)
		optic disc area	mm 2 )	2.23 mm 2 )	mm 2 )
	VCC	NFI	18.6 (±10.7)	16.3 (±9.1)	14.1 (±6.0)	0.179
		TSNIT average	55.3 (±6.3)	55.6 (±5.8)	56.9 (±3.4)	0.506
		Superior average	66.5 (±8.5)	67.7 (±10.7)	70.0 (±6.0)	0.345
		Inferior average	66.5 (±10.3)	67.1 (±8.5)	67.7 (±6.5)	0.870
		TSNIT SD	24.5 (±5.4)	26.1 (±4.7)	25.7 (±4.6)	0.456
		TSS	96.4 (±6.7)	97.3 (±3.9)	94.5 (±7.0)	0.230
	ECC	NFI	20.3 (±8.6)	17.1 (±10.1)	15.8 (±6.4)	0.157
		TSNIT average	51.6 (±5.1)	54.6 (±6.4)	54.5 (±4.3)	0.076
		Superior average	64.0 (±7.7)	66.3 (±11.0)	66.6 (±6.4)	0.500
		Inferior average	65.6 (±9.0)	69.3 (±8.5)	68.8 (±6.9)	0.208
		TSNIT SD	26.9 (±4.3)	28.3 (±4.4)	27.8 (±3.9)	0.498
		TSS	99.7 (±1.0)	98.6 (±4.2)	98.2 (±6.4)	0.420
	*

Influence of optic disc size on parameters of retinal nerve fiber analysis as measured using GDx VCC and ECC in healthy subjects.

noise. By introducing and later removing a preset measurement bias, the instrument's measurement sensitivity is enhanced, what reduces its susceptibility to such errors. This could explain why atypical retardation is reduced with ECC. In the present study our data confirm the previous finding of Toth et al. [START_REF] Toth | Enhanced corneal compensation for scanning laser polarimetry on eyes with atypical polarisation pattern[END_REF] Although the TSS scores were very high in both groups we were able to prove a significant difference between the VCC and the ECC derived values (p<0.001, Wilcoxon test).

Concerning the influence of optic disc size on disc morphology our findings are consistent with literature. All HRT3 parameters of areas and volumes within the optic disc were highly significantly dependent on optic disc area as measured with HRT3. [START_REF] Hoffmann | Optic disk size and glaucoma[END_REF] We could not find a significant impact of optic disc size on any retinal nerve fiber parameter measured with GDx VCC or ECC and with HRT3. In contrast, all parameters of optic disc morphology measured with HRT3, showed statistical significance between the three groups of different optic disc areas. The size of the optic disc does not appear to be an important factor to be taken into account, when analysing images of the GDx VCC or ECC.

Licence for Publication

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Competing Interest

None declared.


MD Andrzej Stankiewicz

MD Joanna Wierzbowska
email: joanna.wierzbowska@gmail.com

Anna Siemi¹tkowska

Beata Fuksiñska

MD Jacek Robaszkiewicz

Arkadiusz Zegad

MD Rita Ehrlich

PhD Brent Siesky

MS Alon Harris

MD Anna Siemiątkowska

MD Beata Fuksińska

MD Arkadiusz Zegadło

The Additive Effect of Dorzolamide Hydrochloride 2% to Bimatoprost 0,03% on Intraocular Pressure and Retrobulbar Blood Flow in Patients with Primary Open-Angle Glaucoma

Keywords: primary open angle glaucoma, intraocular pressure, color Doppler imaging, ocular perfusion pressure

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

INTRODUCTION

The pathogenesis of glaucoma can involve both mechanical and vascular factors, either independently or by influencing one another. Ocular ischemia, induced primarily by vasospasm and autoregulatory dysfunction intensified by elevated intraocular pressure (IOP) may trigger glutamate-mediated toxicity and neurotrophic factor withdrawal and contribute to apoptotic retinal ganglion cell death. [START_REF] Osborne | Ganglion cell death in glaucoma: what do we really know?[END_REF] Numerous studies have shown evidence of systemic circulatory disturbances, perfusion deficits in cerebral and reduced flow in retrobulbar and ocular vessels in glaucoma patients. [START_REF] Graham | Ambulatory blood pressure monitoring in glaucoma. The nocturnal dip[END_REF][START_REF] Harris | Nocturnal ophthalmic arterial hemodynamics in primary open-angle glaucoma[END_REF] Lowering of IOP remains the only current therapeutical approach for preserving visual function in patients suffering from glaucoma and is the goal of currently available glaucoma treatment. It is well established that some glaucoma patients benefit from achieving low target IOP. [START_REF] Leske | Factors for glaucoma progression and the effect of treatment: the early manifest glaucoma trial[END_REF] Multiple medical therapies are often required to achieve such IOP levels. The efficacy of antiglaucoma therapy should be assessed not only on the bases of one measurement of IOP during office hours but also on the basis of diurnal IOP curves. Large diurnal fluctuations in IOP have been shown to be associated with deterioration of visual field in glaucoma patients [START_REF] Asrani | Large diurnal fluctuations in intraocular pressure are an independent risk factor in patients with glaucoma[END_REF] and IOP fluctuations have been identified outside office hours. [START_REF] Barkana | Clinical utility of intraocular pressure monitoring outside of normal office hours in patients with glaucoma[END_REF] Some glaucoma patients continue to progress despite well-controlled IOP. A number of studies report relationship between ocular blood flow deficits and glaucoma progression [START_REF] Zeitz | Glaucoma progression is associated with decreased blood flow velocities in the short posterior ciliary artery[END_REF] or severity. [START_REF] Plange | Asymmetric visual field loss and retrobulbar haemodynamics in primary open-angle glaucoma[END_REF] Improvement of ocular and retrobulbar perfusion may be effective in preventing visual field progression in glaucoma patients.

Nocturnally induced lowering of arterial blood pressure [ABP], caused by a reduction in sympathetic activity during the night, leads to a decrease in heart rate and cardiac input and, when coupled with a concurrent increase in IOP may impair the ocular perfusion pressure (OPP) and blood flow. It has been reported that glaucoma patients with greater nocturnal ABP reduction demonstrated greater progression of optic nerve damage. [START_REF] Graham | Ambulatory blood pressure monitoring in glaucoma. The nocturnal dip[END_REF] Prostaglandin analogs (PGAs) and prostamides are potent IOP lowering agents widely used in glaucoma treatment. PGAs and prostamides reduce IOP to a similar extent during the night and day. [START_REF] Quaranta | Effects of Topical Hypotensive Drugs on circadian IOP, Blood Pressure and Calculated Diastolic Ocular Perfusion Pressure in Patients with Glaucoma[END_REF] Some studies [START_REF] Orzalesi | Comparison of the effects of latanoprost, travoprost, and bimatoprost on circadian intraocular pressure in patients with glaucoma or ocular hypertension[END_REF] suggest that evening-dose PGAs provided better IOP control in the daytime than in the nighttime hours. The hemodynamic characteristic of PGAs is uncertain.

Topical carbonic anhydrase inhibitors (TCAI) are highly effective at night. [START_REF] Quaranta | Effects of Topical Hypotensive Drugs on circadian IOP, Blood Pressure and Calculated Diastolic Ocular Perfusion Pressure in Patients with Glaucoma[END_REF] Dorzolamide, the first TCAI available on the market, has been repeatedly shown to have a positive impact on ocular hemodynamics. [START_REF] Arend | Evaluation of retinal haemodynamics and retinal function after application of dorzolamide, timolol and latanoprost in newly diagnosed open-angle glaucoma patients[END_REF] Combination of prostamides and TCAI, two drugs with different mechanism of hypotensive action, may result in an additive effect on the reduction of IOP [START_REF] Kimal | Additive effect of latanoprost and dorzolamide in patients with elevated intraocular pressure[END_REF][START_REF] Maruyama | Additive effect of dorzolamide or carteolol to latanoprost in primary open-angle glaucoma: a prospective randomized crossover trial[END_REF] and a change in ocular blood flow.

The purpose of the study was to examine the additive effect of dorzolamide hydrochloride to bimatoprost on both diurnal IOP curve and retrobulbar hemodynamics in POAG patients.

MATERIALS AND METHODS

The study was performed according to the tenets of the Declaration of Helsinki and was approved by an institutional review board at the Military Health Service Institute, in Warsaw. Signed informed consent form was obtained from all patients before study enrollment.

Twenty six [START_REF] Galassi | Ocular hemodynamics and glaucoma prognosis: a color Doppler imaging study[END_REF] patients (aged 60.3±10.4 years, range 35-80; 20 women) with a diagnosis of early to moderate POAG under prostaglandin analogs or prostamides monotherapy, were initially enrolled in an open label, prospective, interventional, singlemasked study. After 1-week of run-in period with bimatoprost 0.03% all patients were treated with bimatoprost 0.03% dosed once daily in the morning (9 AM) for 1 month, after which dorzolamide hydrochloride 2% was added twice daily (8 AM and 8 PM) for 2 months. One patient was excluded from the study because of dorzolamide intolerance.

POAG was diagnosed on the basis of the presence of typical glaucomatous visual field changes in two consecutive automated static threshold perimetry (abnormal glaucoma hemifield test or cluster of three non-edge contiguous points at the <5% level on the pattern deviation plot with at least one point at the <1% level or corrected pattern deviation (CPSD) < 5%, with ≤ 25% fixation losses and/or false positives and/or false negatives responses).

Patients of either sex, aged ≥ 18 years with bilateral POAG, without significant cardiac or pulmonary conditions were eligible for the study. Subjects were instructed to avoid caffeine, alcohol, smoking and exercise for 24 hours before being admitted to the hospital and on the study day.

Patients with ocular hypertension, secondary glaucoma, history of intraocular surgery, trauma, inflammation, diabetic retinopathy or other progressive retinal disease, myopia above -6,0 D, CCT < 500 nm and > 600 nm were excluded from the study as were pregnant or nursing women.

After 1-week of run-in period with bimatoprost 0.03% (baseline visit) best corrected visual acuity (BCVA), Goldmann applanation tonometry (GAT), slit-lamp and stereo optic disc evaluation, heart rate (HR) (radial pulse) and systolic and diastolic brachial blood pressure (BP) measurement (sphygmomanometry) were performed. After one month of bimatoprost therapy (control visit) and after two months of combined bimatoprost/dorzolamide therapy (final visit) all patients were admitted to the hospital for 24hours. Goldmann IOP, BP and HR measurements were taken every 2 hours and CDI measurements were taken 5 times over 24 hours. Ocular hemodynamics were assessed by CDI, using LOGIQ 7 Color Doppler Imaging System (General Electric Medical Systems, Milwaukee, Wisconsin, USA) with a 6.7 MHz linear probe and was carried out at 6 PM, 10 PM, 2 AM, 4 AM and 6 AM, by the same investigator, whom the individual therapy of the patients had been masked from. The measurements technique is described elsewhere. [START_REF] Harris | Test/retest reproducibility of color Doppler imaging assessment of blood flow velocity in orbital vessels[END_REF] Peak systolic (PSV) and end-diastolic velocities (EDV) were measured in the ophthalmic (OA) and central retinal arteries (CRA) and Pourcelot's resistance index (RI) was calculated for each vessel.

Mean systolic (SBP) and diastolic (DBP) blood pressures were calculated as an average of all diurnal SBP and DBP readings. Mean arterial pressure (MAP) was calculated as follows: DBP + 1/3 (SBP -DBP). Systolic ocular perfusion pressure (SOPP) and diastolic ocular perfusion pressure (DOPP) were defined as the difference between SBP and IOP, and DBP and IOP, respectively. Mean ocular perfusion pressure (MOPP) was calculated as follows: 2/3 MAP -IOP.

One study eye was chosen randomly for analysis. Twelve right eyes and thirteen left eyes were finally analyzed. In an a-priori-power-analysis the sample size was calculated. With an n-value of 25 we have at least 80% power to detect differences of at least 20% at an alpha level of 0.05. Statistical analysis of the data was carried out using SPSS 10.0 software. The comparisons of the values between the two phases of treatment were carried out using paired t-test and repeated-measure ANOVA with the Bonferroni's adjustment for multiple comparisons. P -values <0.05 were considered statistically significant.

RESULTS

Twenty five patients (aged 61.5 ± 10.9 years, range 35-80; 20 women) completed the entire study. The patient characteristics are presented in the Table 1. 1.

The systolic-diastolic blood pressure (SBP, DBP), MPP and HR between two phases of treatment were evaluated and no statistically significant differences were demonstrated (Table 2). No change in BCVA was found between study visits.

DISCUSSION

To our knowledge this study represents the first prospective clinical trial to evaluate the impact of dorzolamide in addition to bimatoprost therapy in POAG patients on both 24hour IOP curve and retrobulbar hemodynamics.

Numerous studies have shown augmented IOP-lowering effect when dorzolamide was added to prostaglandin analogs (PGAs) or beta-blockers. [START_REF] Kimal | Additive effect of latanoprost and dorzolamide in patients with elevated intraocular pressure[END_REF][START_REF] Maruyama | Additive effect of dorzolamide or carteolol to latanoprost in primary open-angle glaucoma: a prospective randomized crossover trial[END_REF] We hypothesized that the combination of two hypotensive drugs, each with a different mechanism of action would induce an additive effect on the reduction of IOP. The published literature lacks information on hypotensive effect of such a combination. Our study did no show additional lowering effect when dorzolamide was added to bimatoprost at all time points, except at 4 AM. It is possible that relatively low IOP achieved on bimatoprost monotherapy may have contributed to the lack of significant decrease in IOP after adding the dorzolamide. The results obtained by Maruyama [START_REF] Maruyama | Additive effect of dorzolamide or carteolol to latanoprost in primary open-angle glaucoma: a prospective randomized crossover trial[END_REF] with dorzolamide added to latanoprost, confirms our observation. The addition of dorzolamide to latanoprost reduced IOP from 16.0 ± 2.1 mm Hg at the end of latanoprost monotherapy to 15.0 ± 1.3 mm Hg, resulting in only additional mean IOP reduction of 5.6%. They also had a higher (19.1 ± 1.9 mm Hg) baseline IOP compared to our study. These observations are also in agreement with other studies which found a smaller IOP-lowering effect of antiglaucoma medications in normal tension glaucoma patients. [START_REF] Nakamoto | Effect of concomitant use of latanoprost and brinzolamide on 24-hour variation of IOP in normal-tension glaucoma[END_REF] In NTG patients, bimatoprost produces a 19.9% reduction in baseline IOP [START_REF] Dirks | A 3-month clinical trial comparing the IOPlowering efficacy of bimatoprost and latanoprost in patients with normal-tension glaucoma[END_REF] whereas in patients with PEX glaucoma, its efficacy doubles (35%). [START_REF] Konstas | Diurnal IOP control with bimatoprost versus latanoprost in exfoliative glaucoma: a crossover, observer-masked, three-centre study[END_REF] Arici [START_REF] Kimal | Additive effect of latanoprost and dorzolamide in patients with elevated intraocular pressure[END_REF] showed that dorzolamide could be successfully combined with latanoprost to reduce IOP in patients with high-pressure glaucoma (at mean baseline IOP of 26.8 mmHg), resulting in an additional 15% reduction of IOP (15.9 to 13.1 mmHg).

The results of our study demonstrated that bimatoprost/dorzolamide combined therapy provided significantly more stable diurnal and nocturnal IOP curve than bimatoprost monotherapy. The 24-hour IOP fluctuations were 4.6 and 6.0 mmHg, respectively. The lowest value of IOP was observed on bimatoprost monotherapy, starting 12 hours after drug administration. This result is in an agreement with other studies on the 24-hour effect of bimatoprost on IOP. [START_REF] Parrish | A comparison of latanoprost, bimatoprost, and travoprost in patients with elevated intraocular pressure: a 12-week, randomized, masked-evaluator multicenter study[END_REF] In our study, dorzolamide, added to bimatoprost sustained the lowest value of IOP from 10 PM to 2 AM (p=ns) and produced further IOP reduction at 4 AM (p=0.013) and 6 AM (p=ns). In another study [START_REF] Orzalesi | Effect of timolol, latanoprost, and dorzolamide on circadian IOP in glaucoma or ocular hypertension[END_REF] dorzolamide has also demonstrated lower nighttime pressures than daytime ones. Nakamoto [START_REF] Nakamoto | Effect of concomitant use of latanoprost and brinzolamide on 24-hour variation of IOP in normal-tension glaucoma[END_REF] however showed greater additive effect of brinzolamide when was added to latanoprost in daytime (5.7%) than in nighttime (3.4%).

Addition of dorzolamide to bimatoprost did not cause any statistically significant differences in the systolic-and diastolic arterial blood pressure, systolic-and diastolic ocular perfusion pressure and heart rate. Our results are in an agreement with a previous study. [START_REF] Siesky | A comparative analysis of the effects of the fixed combination of timolol and dorzolamide versus latanoprost plus timolol on ocular hemodynamics and visual function in patients with primary open-angle glaucoma[END_REF] Bimatoprost is a derivative of the prostamide and its ocular action is similar to PGF 2α analogs, latanoprost and travoprost. [START_REF] Lim | Mechanism of action of bimatoprost, latanoprost, and travoprost in healthy subjects. A crossover study[END_REF] The studies examining the effect of latanoprost [START_REF] Arend | Evaluation of retinal haemodynamics and retinal function after application of dorzolamide, timolol and latanoprost in newly diagnosed open-angle glaucoma patients[END_REF] or bimatoprost [START_REF] Zeitz | Ocular hemodynamics in normal tension glaucoma: effect of bimatoprost[END_REF] on retrobulbar blood flow showed no hemodynamic effect both for PGAs and prostamides.

In our study in patients with POAG, treated with bimatoprost, the addition of dorzolamide had small measurable effect on the retrobulbar vasculature. Dorzolamide led to a significant decrease of RI in OA while the other parameters remained stable.

Some authors found an increase in retrobulbar velocities and decrease of resistance index after topically applied dorzolamide or dorzolamide/timolol fixed combination [START_REF] Siesky | A comparative analysis of the effects of the fixed combination of timolol and dorzolamide versus latanoprost plus timolol on ocular hemodynamics and visual function in patients with primary open-angle glaucoma[END_REF],

whereas others reported lack of any flow changes in the retrobulbar vessels. [START_REF] Harris | Effect of dorzolamide timolol combination versus timolol 0.5% on ocular bloodflow in patients with primary openangle glaucoma[END_REF] The OA, unlike the CRA and short posterior ciliary arteries (SPCA) is a large caliber vessel, relatively resistant to changes in IOP. [START_REF] Harris | Acute IOP elevation with scleral suction: effects on retrobulbar haemodynamics[END_REF] Lack of flow velocity changes in this large vessel in our study is not surprising. Parallel changes in both PSV and EDV of the OA have rarely been observed. In healthy subjects flow velocities were reduced in response to hypoxia.

Hypoxia did not induce PSV and EDV changes of OA in glaucoma patients, possibly because of preexisting vasospasm. Increase of EDV and decrease of RI in the OA were reported in NTG patients in response to hypercapnia, which also supports the hypothesis of preexisting vasospasm, partially reversed under hypercapnia. [START_REF] Hosking | Ocular haemodynamic responses to induced hypercapnia and hyperoxia in glaucoma[END_REF] Systemic CAI did not alter PSV and EDV of OA in young healthy volunteers, while their action in glaucoma patients has not been studied. The hypothesis that topically applied CAI may regulate the vascular spasm of OA in glaucoma patients and decrease the flow resistance requires further investigation. In our study we observed significantly decrease of RI in OA after adding of dorzolamide to bimatoprost.

Gallasi et al. [START_REF] Galassi | Ocular hemodynamics and glaucoma prognosis: a color Doppler imaging study[END_REF] reported that increased RI in OA was associated with an approximately 6fold increase of the risk of visual field deterioration, suggesting that RI changes at the level of OA may have valuable prognostic value.

We did not observe any significant changes in PSV, EDV or RI of CRA. The CRA, as a smaller caliber vessel is more sensitive to fluctuations in perfusion pressure, mostly due to IOP change. [START_REF] Strenn | Reproducibility and sensitivity of scanning laser Doppler flowmetry during graded changes in PO 2[END_REF] Lack of significant modification of flow velocities in CRA may be caused both by no change in IOP (and perfusion pressure) after adding of dorzolamide and by low baseline IOP. Siesky [START_REF] Siesky | A comparative analysis of the effects of the fixed combination of timolol and dorzolamide versus latanoprost plus timolol on ocular hemodynamics and visual function in patients with primary open-angle glaucoma[END_REF] and co-workers found that four weeks dorzolamide/timolol fixed combination might increase blood velocities and decrease resistance index in CRA and temporal short posterior ciliary arteries. In another study, Harris et al. [START_REF] Harris | Effect of dorzolamide timolol combination versus timolol 0.5% on ocular bloodflow in patients with primary openangle glaucoma[END_REF] found that addition of dorzolamide to timolol in fixed combination had no measurable effect in the retrobulbar vasculature. The differences in reported hemodynamic outcomes might be due to different baseline IOP levels, 19.1 mmHg [START_REF] Siesky | A comparative analysis of the effects of the fixed combination of timolol and dorzolamide versus latanoprost plus timolol on ocular hemodynamics and visual function in patients with primary open-angle glaucoma[END_REF] compared to 14.7 mmHg in Harris [START_REF] Harris | Effect of dorzolamide timolol combination versus timolol 0.5% on ocular bloodflow in patients with primary openangle glaucoma[END_REF] study and the range of IOP reduction (12% vs. 8%, respectively). It remained to be proven if the concomitant increase in PSV and EDV of CRA relates to increase in volumetric blood flow and how is it related to reduction in IOP.

Ocular blood flow improvement may be partially caused by reduction in IOP, clinical studies also speculated the presence of another, un-known mechanism, independent of the IOP-lowering effect. Fuchsjager-Mayrl [START_REF] Fuchsjäger-Mayrl | Effect of dorzolamide and timolol on ocular blood flow in patients with primary open angle glaucoma and ocular hypertension[END_REF] observed an increase of blood flow in temporal neuroretinal rim, cup disc and increased fundus pulsation amplitude in eyes treated with dorzolamide whereas these changes were not reported in eyes treated with timolol, although it induced a comparable or even greater reduction in IOP and a comparable increase in ocular perfusion pressure.

In summary, in the current study we found that the addition of dorzolamide hydrochloride 2% to bimatoprost 0.03% had an additive significant hypotensive effect on the nighttime IOP curve at 4 AM but did not have on the daytime IOP curve. Dorzolamide added to bimatoprost caused a lower 24-hour IOP fluctuation. Addition of dorzolamide to bimatoprost resulted in significant decrease of resistance index in the OA. Our results suggest that dorzolamide might improve ocular blood flow independently of IOP reduction but the mechanism, by which topical carbonic anhydrase inhibitors influence the vascular bed, remains unknown.

LEGENDS FOR FIGURE AND TABLES

Figure 1 .

 1 Figure 1. Mean intraocular pressure (IOP) readings over 24-hour monitoring; Bimatoprost monotherapy compared to bimatoprost/dorzolamide therapy.

TABLE 1 .

 1 Characteristics of patients Dorzolamide did not lead to a significant change in IOP after two-months of combined therapy (12.2 ± 2.6 mmHg, p=0.188). When pressures were compared at individual time points, there was only one point at 4 AM at which IOP control was significantly improved using the bimatoprost/dorzolamide combined therapy (p=0.013). The greatest IOP reduction under combined therapy was observed at 10 AM, 4 PM and during nighttime at 4

	Variable	Mean	(SD)	Range
	Age (years)	61.5	10.9	35.0-80.0
	IOP (mmHg)	14.8	3.5	11.0-24.0

AM and at 6 AM. The 24-hour IOP fluctuations, defined as a difference between maximum and minimum of IOP were 6.0 ± 2.3 mmHg on bimatoprost monotherapy and 4.6 ± 1.5 mmHg on bimatoprost/dorzolamide combined therapy (p=0.0016). The mean IOP at each time point after bimatoprost and bimatoprost/dorzolamide combination treatment are provided in Figure

TABLE 2 .

 2 The mean IOP, BP, OPP and HR on Bimatoprost and Bimatoprost + Dorzolamide treatmentThe differences observed in PSV, EDV and RI in OA and CRA before and after adding of dorzolamide are provided in Table3 and Table 4. Dorzolamide caused statistically significant decreasing in mean RI of the OA (p=0.017). All other parameters assessed by CDI measurements in the CRA and OA appeared to be stable, although the tendency of increasing

	Bimatoprost	Bimatoprost + Dorzolamide	p value

TABLE 1 .

 1 Characteristics of patients

TABLE 2 .

 2 The mean IOP, BP, OPP and HR on Bimatoprost and Bimatoprost + Dorzolamide treatment

TABLE 3 .

 3 Ophthalmic artery Hemodynamics by Color Doppler Imaging

TABLE 4 .

 4 Central retinal artery Hemodynamics by Color Doppler Imaging

Acknowledgements

The authors would like to gratefully acknowledge Dr Janusz Sierdziński for his statistical analysis in this study.

Funding for the study was provided by Merck & Co., Inc. and unrestricted grant from research to prevent blindness.


Ailsa E Ritchie

Philip G Griffiths+

Patrick F Chinnery%

Adrian W Davidson^
email: adrian.davidson@nuth.nhs.uk

Eye movement recordings to investigate a supranuclear component in CPEO: a cross sectional study

Keywords:

Background: It has been postulated that eye movement disorders in CPEO have a neurological as well as a myopathic component to them. Aim: To investigate whether there is a supranuclear component to eye movement disorders in CPEO using eye movement recordings. Methods: We measured saccade and smooth pursuit characteristics together with vestibulo-ocular reflex (VOR) gain and VOR suppression (VORS) gain in 18 patients with CPEO and 34 normals using Eyelink II video-oculography.

Results:

The asymptotic values of the peak velocity main sequence curves were reduced in the CPEO group compared to normals, with a mean of 161 deg/sec (95% CI 126-197 deg/sec) compared to 453 deg/sec (95% CI 430-475 deg/sec) respectively. Saccadic latency was longer in CPEO (263ms; CI 250-278), compared to controls (185ms; CI 181-189). Smooth pursuit and VOR gains were impaired in CPEO although this could be explained by non-supranuclear causes. VORS gain was identical in the two groups. Conclusions: This study does not support a supranuclear component to the ophthalmoplegia of CPEO, although the increased latencies observed may warrant further investigation.

INTRODUCTION

Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial disorder characterised by progressive restriction of eye movements, ptosis and orbicularis weakness. Although myopathy accounts for a major part of the restriction of eye movements seen in CPEO some authors have suggested that there may be a supranuclear component to the ophthalmoplegia. [START_REF] Gupta | Supranuclear eye movement dysfunction in mitochondrial myopathy with tRNA(LEU) mutation[END_REF] The distinction between myopathic and supranuclear components is not purely academic because the success of strategies to regenerate normal extraocular muscle, by stimulating endogenous satellite cells, [START_REF] Andrews | Evaluation of bupivacaineinduced muscle regeneration in the treatment of ptosis in patients with chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome[END_REF] or by using embryonic stem cells, is dependent on CPEO being primarily a myopathy. Suppression of the vestibulo-ocular reflex (VORS) measures central aspects of eye movement control since if VOR suppression is complete no actual eye movement has to be generated, thus isolating out the myopathic component of the eye movement problem. Impaired suppression of VOR has been cited as evidence of supranuclear involvement in myotonic dystrophy. [START_REF] Anastasopoulos | Abnormalities of ocular motility in myotonic dystrophy[END_REF][START_REF] Kimmig | Role of anterior and occipital white matter lesions for smooth eye tracking in myotonic dystrophy[END_REF]

METHODS

Ethical approval was gained from the local research ethics committee before starting the study. Patients were recruited into the study if they had a slowly progressive ophthalmoplegia, cytochrome c oxidase negative fibres on muscle biopsy and had given written, informed consent.

Eye movement recordings

Eye movements were recorded using an EyelinkII video-oculography system (SR Research, Canada) at 500 samples per second. Calibration, saccadic and smooth pursuit stimuli were produced at a distance of approximately 1100mm from the subject. The distance was measured and stimulus angle calculated individually for each subject.

All tests were done with the subject seated in a MiniTorque Barany chair (Difra, Belgium). A chinrest was mounted on the chair, which substantially reduced but did not completely eliminate head movements. Eyelink long-range infra-red markers for head movement compensation were mounted on the fixation board and used during the Eyelink internal calibration. Eyelink head referenced data was used in the analysis and the head movement markers were turned off during VOR testing. Saccades and smooth pursuit were recorded in low level lighting, and VOR was measured in total darkness. Eyelink calibration was done at an angle suitable for the range of movements of the subjects' eyes. This ranged between +/-7 degrees and +/-26 degrees horizontally and +/-5 and +/-20 degrees vertically, using a 5 point calibration. Some subjects had ptosis, in which case the eyelids were taped up. Where the ptosis still interfered with calibration a horizontal 3 point calibration was employed. The best possible Eyelink calibration was obtained, but in addition, up to 9 horizontal fixations were used to derive a third order polynomial which was retrospectively used as a secondary calibration and applied to the horizontal signals. Where the subject had limited eye movements, only those fixations within the range of valid movements were used for secondary calibration. Subjects with tropias were calibrated monocularly. Ideally, all subjects should have been calibrated monocularly. Calibration was difficult for some subjects in the CPEO group.

Saccades

Saccades were measured using an LED board. A zero-gap paradigm was used with stimulus amplitude up to a maximum of 26 deg left or right. Direction and timing were randomized. Leftward and rightward primary centrifugal saccades were used in the analysis. The start and end of saccades was defined at the points where velocity dropped below 20 deg/s. We excluded saccades contaminated by blinks, artefacts or with a latency less than 100ms.

Smooth pursuit

Smooth pursuit was generated with a laser spot projected via a galvanometer mirror, with a sinusoidal profile of +/-10 degrees at 0.4, 0.6 and 0.8 Hz (peak velocities of 25, 38 and 50 deg/s), the amplitude being chosen with the expectation that most CPEO patients would have that degree of eye movement.

VOR and VOR suppression

VOR was generated using the Barany chair, using sinusoidal stimulation of +/-80 deg at 0.1 and 0.2Hz (peak velocities of 50 and 100 deg/s). Subjects were asked to imagine viewing a distant object and to perform mental arithmetic to maintain alertness. The VOR suppression stimulus was an LED mounted on a moveable stalk, positioned in front of the subject at a distance of approximately 50 to 60cm, depending on the size of the subject.

Statistical analysis

For each subject, only the "best eye" data was used, and this was determined from consideration of the Eyelink calibration reports and subjective examination of the quality of the signals.

Peak saccadic velocity main sequence curves were derived for each subject by fitting data to an equation of the form

PV = PVmax * (1-e-A/c)
where PVmax is the asymptotic value of the curve, A is the amplitude of the saccade, and C is a constant relating to the slope. The mean and 95% confidence interval (CI) of the mean were then derived for PVmax.

Saccade latencies from all studies were pooled for the control and CPEO groups. Only the first 13 primary saccades for each study were used, to ensure that the mean was not biased due to uneven numbers of saccades per study. Because the distribution of latencies was skewed, the reciprocal of the latencies were taken, mean and CI were calculated, and the values transformed back. [START_REF] Leigh | The Neurology of Eye Movements[END_REF] We hypothesised that VORS gain may be related to VOR gain such that subjects with a lower VOR gain would find it easier to suppress their VOR, and therefore would be more likely to have a lower VORS gain. We plotted VORS gain against VOR gain, to see if there was any relationship.

If the VOR was perfect, that is, the countermovement of the eyes perfectly matched the rotation of the chair we would expect a VOR gain of one. If VORS was perfect we would expect a gain during chair rotation, while fixating the suppression LED, of zero.

RESULTS

18 patients with CPEO were tested (median age 59 years). All patients had cytochrome c oxidase negative fibres in skeletal muscle biopsies and slowly progressive ophthalmoplegia. 7 patients had multiple deletions, 7 had single deletions, 2 had point mutations and in 2 cases no mutation could be identified. We could not reliably perform all of the measurements on all of these patients, and therefore the numbers for the different measurements differ slightly. Thirty four control patients were tested (median age 36 years).

Saccades

As expected, peak saccadic velocity was slow in CPEO in comparison with controls. Figure 1 shows the peak velocity for each saccade plotted against its amplitude (the peak velocity main sequence). The mean asymptotic peak velocity (PVmax) was 453 deg/s for controls (CI 430-475) and 161 deg/s for CPEO .

For analysis of latency we used the same number of saccades from each subject in order to avoid biasing the results. Studies with a minimum of 13 primary centrifugal saccades suitable for measuring latency were used and the results are shown in figure 2.

Mean saccadic latency in CPEO (263ms, CI 250-278) was increased in relation to controls (185, CI 181-189), although many saccades made by patients with CPEO had short latency times which overlapped with those of the control group.

Smooth pursuit

Participants who were unable to maintain smooth pursuit at all frequencies were excluded. Some controls had measurements made at different frequencies during the early stages of data collection and their data is excluded from the statistical calculations, however, the measurements at these frequencies are plotted on Figure 3. There was some impairment of smooth pursuit in patients with CPEO compared to normal controls at all frequencies. At 0.4Hz mean gain for controls was 0.89 (0.85-0.92) compared to 0.71 (0.59-0.83) in the CPEO group. At 0.6Hz the means were 0.80 (0.72-0.89) in controls and 0.53 (0.39-0.66) for CPEO. Finally, at 0.8Hz the control group mean was 0.65 (0.54-0.77) compared to 0.37 (0.25-0.49) in CPEO.

VOR and VORS

VOR gain was lower in the CPEO group than in controls. At 0.1Hz, mean VOR gain was 0.65 (0.58-0.72) in the control group and 0.48 (0.39-0.57) in the CPEO group. At 0.2Hz, mean VOR gain was 0.61 (0.55-0.67) in controls and 0.34 (0.25-0.44) in the CPEO group. Means and 95% confidence intervals were almost identical for VORS gains. At 0.1Hz both groups had a mean of 0.06, but the CPEO group had a slightly larger confidence interval of 0.05-0.08 compared to 0.05-0.07 for controls. At 0.2Hz both means were 0.09, with a control CI of 0.07-0.12 and a CPEO CI of 0.07-0.11 . Figure 4 shows that there is no relationship between VOR gain and VORS gain.

DISCUSSION

Interpretation of results should always take account of the limitations of the measurements. In our case this mainly concerns the inherent inaccuracy of subject fixation, small head movements, and potential errors in calibration. These will introduce some variability into the measurements.

There is no doubt that there is a major myopathic component to CPEO. Evidence for this includes loss of muscle bulk on MRI scans, [START_REF] Carlow | MR of extraocular muscles in chronic progressive external ophthalmoplegia[END_REF] findings on muscle biopsy [START_REF] Ringel | Extraocular muscle biopsy in chronic progressive external ophthalmoplegia[END_REF] and the appearance of muscles at surgery. It has been proposed that eye muscles are uniquely vulnerable in mitochondrial myopathies because of their functional and ultrastructural properties. [START_REF] Wai Man | Extraocular muscles have fundamentally distinct properties that make them selectively vulnerable to certain disorders[END_REF] However, these findings do not preclude an underlying supranuclear component. Some previous studies point in this direction. There have been isolated case reports of patients with a greater range of eye movements to oculocephalic testing than ductions. [START_REF] Gupta | Supranuclear eye movement dysfunction in mitochondrial myopathy with tRNA(LEU) mutation[END_REF] Magnetic resonance imaging has shown abnormal metabolic profiles of the brain in patients with mitochondrial myopathies including CPEO [START_REF] Salvan | Brain metabolic profiles obtained by proton MRS in two forms of mitochondriopathies: Leber's hereditary optic neuropathy and chronic progressive external ophthalmoplegia[END_REF] and brain stem reflexes have been found to be abnormal in patients with mitochondrial myopathies. [START_REF] Koutroumanidis | Reduced brain stem excitability in mitochondrial myopathy: evidence for early detection with blink reflex habituation studies[END_REF] The observed reduced saccadic velocities in CPEO are well documented [START_REF] Yee | Saccadic eye movements in myasthenia gravis[END_REF] and most likely muscular in origin. Increased latency might be taken as evidence of supranuclear pathway dysfunction, so that it takes longer to generate the pulse required to move the eyes. Latency is easier to measure reliably in CPEO subjects than other parameters, since it is less influenced by calibration errors and ptosis. However, we hesitate to argue for abnormal supranuclear function from this data alone, since other factors may be involved. One factor is the difference in age distribution of the two groups, although this is unlikely to account for all the difference. Irving et al [START_REF] Irving | Horizontal saccade dynamics across the human life span[END_REF] found that subjects below the age of 50 had mean latencies of around 195ms, and this rose in each decade to around 265ms in those 80 or older. Using the mean values from their chart, we estimated the effect on our group means that each subject would have made due to the age difference with a nominal reference age. Depending on the reference age, we could account for 25 to 30ms of the observed 78ms difference in means between the groups.

VOR is responsible for maintaining images steady upon the retina while the head moves. It is a brain stem reflex mediated by the vestibular apparatus, vestibular nuclei and oculomotor nuclei. The maintenance of steady gaze while reading in a moving vehicle, for example, requires the ability to suppress the VOR. VORS is mediated by the pursuit pathway [START_REF] Bittencourt | Determination of smooth pursuit eye movement velocity in humans by computer[END_REF][START_REF] Chambers | The relationship between disordered pursuit and vestibuloocular reflex suppression[END_REF] and the cerebellum. [START_REF] Mira | Impaired suppression of vestibular nystagmus by fixation of visual and acoustic targets in neurological patients[END_REF] In the ophthalmoplegia of myotonic dystrophy, parallel impairment of SP and VORS has been taken as evidence of a supranuclear component to the ophthalmoplegia. [START_REF] Anastasopoulos | Abnormalities of ocular motility in myotonic dystrophy[END_REF][START_REF] Kimmig | Role of anterior and occipital white matter lesions for smooth eye tracking in myotonic dystrophy[END_REF] We found impairment of SP (although there is large variability and considerable overlap with controls) and VOR, but no impairment of VORS. If the impaired VOR is due to peripheral ocular muscle weakness, then there would likely have been adaptive changes in the internal VOR gain, which nevertheless may have been insufficient to overcome the peripheral muscle weakness. The closed loop nature of the SP control system, along with parallel adaptation in the SP system, [START_REF] Optican | Adaptive response to ocular muscle weakness in human pursuit and saccadic eye movements[END_REF][START_REF] Carey | Signals that modulate gain control for smooth pursuit eye movements in monkeys[END_REF] may enable continued suppression of the VOR. However, as with the VOR, this may still be insufficient to overcome the peripheral weakness, so that SP is still impaired. This would explain our observations of impaired VOR and SP, but preserved VORS. This suggests a peripheral rather than central pathology

CONCLUSION

While the observed increase in latency is consistent with a possible supranuclear component, there are other potential contributing factors. Although we found that the VOR gain was reduced in CPEO, VORS gain and confidence intervals were almost identical in the two groups. Since VORS is mediated by the pursuit pathway this supports our view that the reduction in smooth pursuit gain in CPEO is not supranuclear in origin. We did not observe any relationship between VORS gain and VOR gain. We considered VORS to be the more robust indicator in subjects with ophthalmoplegia and it therefore seems unlikely from our data that supranuclear dysfunction is the primary event in CPEO. However, the increased latency observed may warrant further investigation.

Because of the small numbers it not possible to make distinctions between CPEO due to single or multiple deletions.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Peak saccadic velocity of primary centrifugal saccades in controls (crosses, n=34) and CPEO patients (circles, n=18)

Figure 4 :

 4 Figure 4: VORS gain vs VOR gain at 0.1Hz (a) and 0.2Hz (b). Controls represented by crosses and CPEO by circles.

  Fig 1

Acknowledgements

PFC is a Wellcome Trust Senior Fellow in Clinical Science who also receives funding from the Medical Research Council (UK), the UK Parkinson's Disease Society, and the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust.

Competing interests: None declared

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