© 2002 American College of Veterinary Ophthalmologists

Veterinary Ophthalmology

(2002)

5

, 1, 19–22

Blackwell Science Ltd

SLO angiography: arterio-venous filling times in monkey and minipig

Serge G. Rosolen,* Gérard Saint-Macary,† Vincent Gautier‡ and Jean-François Le Gargasson‡

*

Clinique vétérinaire, 119 Boulevard Voltaire, 92600 Asnières, France,

†

Synthelabo-Recherche, 2–8 rue de Rouen, ZI Limay Porcheville, 78440 Gargenville, France,

‡

INSERM U-483, 10 avenue de Verdun, 75010 Paris, France

Abstract

Confocal scanning laser ophthalmosocope (cSLO) is a new technique which enables ocular fundus image recording and dynamic retinal angiography to be performed. The ocular fundus image is acquired sequentially, point by point, and is reconstructed on a video monitor at the rate of 25 images per second. The aim of this paper is to evaluate the feasibility of measuring retinal arterio-venous filling times (AVFT) with a I + Tech cSLO. Three young adult cynomolgus monkeys and three young adult Göttingen minipigs were used as experimental models. All animals were anesthetized using a zolazepam + tiletamine mixture injected intramuscularly; heart rate and rectal temperature were monitored and corneal irrigation was regularly performed. For all subjects, prior to examination, hematocrit and globe axial length were measured. The images were recorded, stabilized and analyzed. The retinal examination consisted of retinal images with 40

°

field cSLO, retinal fluorescein angiography and arterio-venous 50% filling time measurements. For each subject all images were easily recorded while keeping the animals in a normally lighted room without having to use any additional optical device. AVFT using an I + Tech cSLO is easily performed in monkeys and minipigs. AVFT measurements in minipigs and monkeys are similar. These results suggest that minipigs can replace monkeys as an experimental species for AVFT investigations.

Key Words:

angiography, minipig, monkey, SLO

Address communications to:

Dr Serge G. Rosolen

Tel.: +33 1 47 33 08 95 Fax: +33 1 47 33 30 37 e-mail: rosolen@club-internet.fr

INTRODUCTION

The confocal scanning laser ophthalmoscope (cSLO) mini-mizes blurring and reflected images and enhances contrastby illuminating only a limited portion of tissue at selecteddepths in the retina. Combined with digital image analysisits applications are multiple in investigations of dynamicretinal function,

1,2

particularly for measurement of earlyarterio-venous filling times (AVFT).

3,4

Many researchers haveevaluated retinal microcirculation in animal models such asmonkeys

5

or pigs by use of fluorescein dye.

6

For many, theadult minipig represents one of the best models to studythe physiology system

7

including retinal blood flow.

8

Indeed, the visual system of the minipig is quite similar tothat of humans.

9,10

The aim of this report is to test thefeasibility of AVFT measurements during early retinal fillingtimes in monkeys and minipigs.

MATERIALS AND METHODS

Our protocol adhered to the Association for Research inVision and Ophthalmology Statement for the Use of

Animals in Ophthalmic and Vision Research, and work wasconducted in Sanofi-Synthelabo-Recherche facilities.

Animals

Three young adult healthy cynomolgus monkeys purchasedfrom CRP (Center de Recherches Primatologiques, Mauri-tius Island) and three young adult healthy Göttingen mini-pigs (Ellegaard Göttingen minipig, Dalmose, Denmark)were included in this study.

All animals were anesthetized with an intramuscular injec-tion of zolazepam + tiletamine (Zoletil

®

, Virbac, Carros, France)mixture at a dose of 20 mg/kg. All had their heads placed ina retention device and clips assured a perfect globe fixity andopening of eyelids. Hydration of the cornea was maintainedduring the examinations using BSS eye drops (Alcon, Rueil-Malmaison, France). Prior to performing the examinations,hematocrit and globe axial length were measured and the pupilswere maximally dilated with drops of tropicamide (Mydriaticum

®

,MSD, Paris, France) in each eye to obtain a stable mydriasisfor at least 1 hour before the recording session. Rectal tem-perature was periodically monitored as well as blood pressureand heart rate (Finapres 2300

®

, Ohmeda, Trappes, France).

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cSLO images

The principle of visualization of the ocular fundus is basedon the use of two-directional deflection of the beam by twooscillating mirrors illuminating, at any one time, a very smallarea of the retina, measuring 10 square micrometers. Thebeam, retrodiffused by the retina, crosses the deflectionmodule and forms a variable flow with fixed direction and isdetected by a photomultiplier; there is a congruent relation-ship between the variation in the flow over time and the zoneof retina from which it is derived. The ocular fundus imageis acquired sequentially, point by point, and is reconstructedon a video monitor at the rate of 25 images per second. Itcan be observed directly or can be recorded by a videotaperecorder for subsequent ‘image by image’ analysis.

11

Theretinal field illuminated instantaneously is very limited andthe interference by light due to corneal reflection and diffu-sion by the ocular media is particularly low.

12

The cSLO (I + Tech, Clermond-Ferrand, France) used inthis study

13

(Fig. 1) is composed of two laser illuminationbeams: an infrared light-emitting diode beam (780 nm) andan argon beam (488 nm) which allows a 40

°

field recordingof retinal images in a lighted environment. These two beamscan be used independently or concomitantly. They may ormay not be modulated by means of an acoustico-optical sys-tem piloted by the video signal of an auxiliary televisioncamera. The argon beam is used for visualization of the

ocular fundus. The energy flow through the pupil of the sub-ject is of the order of 100–300 microWatts for the argonbeam and its maximal luminance (3.100 cd/m

2

) can bereduced by 2- or 3-fold.

Equivalent fundic areas of each monkey and minipig wereexamined without any additional optical devices.

Angiograms

After baseline fundic image recordings, a 10% fluoresceinsolution was injected into the lateral saphenous vein for eachanimal. cSLO recordings commenced at the beginning ofthe injection.

Images analysis

Images were recorded, stabilized and analyzed under the sameconditions. The recorded images (25 frames/s) were analyzedby a personal computer-base image analysis system

14

and NIHimage 1.62 freeware as previously described.

13

The arterial andvenous gray levels were measured on successive stabilizedimages as a function of time and evaluated by visualization ona screen (scale: 256 pixels, acuracy

±

1 pixel). The 100% arte-rial and venous filling times corresponded to the maximum ofwhite level measured for each of them. The intensity of fluo-rescein dye was measured with a photomultiplier. The outputvoltage measurement (intensity as a function of time) was nor-malized to a maximum of 1 (100%) and a minimum of 0.

Figure 1. Diagram of I + Tech confocal scanning laser ophthalmoscope. The ametropies compensator was not used in this study.

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S L O

A N G I O G R A P H Y

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RESULTS

Mean heart rate, globe axial length, hematocrit and AVFTvalues are represented in Table 1. All measured parameterswere stable and constant during each examination. Retinalimages, such as those shown in Fig. 2, were easily recordedwhile the animals were kept in a normally lighted roomwithout having to use any additional optical device.

The AVFT measurements at 50% filling time for mon-keys and minipigs are shown in Fig. 3.

The data presented show a small interindividual variabil-ity as confirmed with the coefficient of variability (C

v

% =standard deviation/mean) which was 5% for the AVFTmeasurements of monkeys and 2% for that of minipigs.

DISCUSSION

There is a little difference between the hematocrit values:monkeys, 44 and minipigs, 42. Despite axial length variationsbetween monkeys and minipigs their AVFT measurements

Heart rate/min Axial length (mm) Hematrocrit (%) AVFT (s)

Monkeys 117 18.2 44.7 2.65122 18.4 44.8 2.95121 18.2 44.7 2.85

Mean ± SD 120 ± 2.65 18.3 ± 0.12 44.7 ± 0.06 2.82 ± 0.15Minipigs 102 23.6 42.1 2.36

109 23.9 42.1 2.44105 23.7 42.2 2.41

Mean ± SD 105 ± 3.5 23.7 ± 0.15 42.1 ± 0.06 2.4 ± 0.04

Table 1 Heart rate, globe axial length, hematrocrit and arterio-venous filling times (AFVT) in monkey (3 animals) and minipig (3 animals). Individual values and mean values.

Figure 2. 40° I + Tech confocal SLO: minipig ocular fundus image (A), fluorescein angiography at 2 s (B), fluorescein angiography at 3 s (C) and fluorescein angiography at 5 s (D) after injection of 10% fluorescein. The 50% AVFT is evaluated by visualization on a screen (scale: 256 pixels, accuracy: ±1 pixel).

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are similar. This suggests that globe size is not a majorparameter when comparing AVFT measurements in mon-keys and minipigs. It should be noted that heart rates weremeasured under anesthesia. These values are lower thanheart rate values in nonanesthetized animals. This suggeststhat heart rate values is linked to AVFT measurement, andthus heart rate should be considered as an important factorwhen performing AVFT investigations. Due to the similar-ity in AVFT results between monkeys and minipigs, oursresults indicate that minipigs can be used as a replacementfor monkeys for AVFT measurements in pharmacologicaland toxicological investigations. However, the replacementof monkeys by minipigs as an ethical consideration is also animportant point.

Fundic images generated with a 40

°

field cSLO wereeasily recorded without any additional optical device beingrequired for each of the species. The high resolution ofimages due to digital analysis makes cSLO a fundamentaltool in the exploration of dynamic retinal microcirculation

in animals for clinical, pharmacological and toxicologicalinvestigations. Studies are in progress in the use of AVFT indogs and cats.

ACKNOWLEDGMENTS

The authors thank Professor Pierre Lachapelle (UniversityMcGill-Hôpital de Montréal pour enfants, Laboratoire dephysiologie Visuelle, Montréal, Québec, Canada) for hishelp with revising this manuscript. The authors also thankthe Société Française d’Etudes et de Recherches en Ophtal-mologie Vétérinaire (S.F.E.R.O.V ), Fondation ophtalmologiqueA. de Rothschild, 25–29 rue Manin, 75019 Paris, France.

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Figure 3. Arterial and venous intensity of fluorescein dye (normalized curves) as a function of time. (A): for the monkey, the arterio venous fill-ing time (AVFT) is 2.95 s . This time represents the difference between the 50% filling time of artery and the 50% filling time of vein. The mean heart rate was 122/min. The hematocrit was 44.8%. The globe axial length was 18.4 mm. (B): for the minipig, the arterio-venous filling time (AVFT) is 2.44 s. This time represents the difference between the 50% filling time of the artery and the 50% filling time of the vein. The minimum value was not equal to zero due to a residual electric offset. The mean heart rate was 109/min. The hematocrit was 42.1%. The globe axial length was 23.9 mm.

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