Ординатура / Офтальмология / Учебные материалы / Uveitis Text and Imaging Text and Imaging Text and Imaging 2009

angiography to analyse the choroidal stromal vasculature, imaging large vessels in the initial angiographic phase.2 Because the machinery was not performing enough they did not look at later angiographic frames where fluorescence was insufficient. From these early publications comes the erroneous concept that indocyanine green is not exudating into the choroidal stroma. It is now clear that even though indocyanine green forms large molecular complexes with proteins, it is massively egressing from the largely fenestrated choriocapillaris to impregnate the choroidal stroma in the intermediate and late phases of angiography remaining during several days thereafter.3 This diffuse stromal low intensity fluorescence can now be detected because, since the introduction of the first digitalised imaging systems much more performing infrared cameras have become available. As a consequence, indocyanine green angiography (ICGA) has been the object of renewed interest because of the good quality and reproducibility of digitalised images allowing their systematic analysis.4,5 Even low fluorescence coming from entraped ICG molecules in the choroidal stroma can be seen in the later angiographic frames. A basic introduction on the principles and interpretation of ICGA in inflammatory diseases is indispensable for a comprehensive appraisal of ICGA semiology and the understanding of angiographic signs.

KEY POINTS: GENERAL ASPECTS

•Fluorescein angiography gives information and is the method of choice to analyse superficial structures of the fundus including optic disc, retina and RPE, as well as the choriocapillaris in the first 60 seconds of the angiogram.

•Indocyanine green angiography is the method of choice to analyse choroidal inflammatory lesions including the choriocapillaris and the choroidal stroma.

•The choroid is the site of origin of the inflammatory process in posterior uveitis at least as often if not more often than the retina and ICGA is indicated whenever choroidal involvement is suspected.

•Early publications on ICGA, done with low performance infrared cameras and limited to the analysis of choroidal stromal vessels showed that ICG remained within the large stromal non inflamed vessels. These studies are at the origin of the erroneous concept that there is no extrusion of ICG into the choroidal stroma.

BASIC PRINCIPLES OF INDOCYANINE GREEN ANGIOGRAPHY

PHARMACOLOGY OF INDOCYANINE GREEN

Indocyanine green is a hydrosoluble molecule with a molecular weight of 775 Da that binds strongly to

Figure 1: ICG molecule

proteins after intravenous injection (Figure 1). The proportion of protein-bound indocyanine green is as high as 98-99% with a large proportion fixed on proteins with a high molecular weight such as globulins (alphalipoproteins).6 The circulating molecule is eliminated through the liver and is found in the bile in a nonmetabolised form. The administration of indocyanine green is usually much better tolerated than the injection of fluorescein that produces quite often a reaction of nausea. However in contrast to fluorescein, indocyanine green contains up to 5% of iodide that can be at the origin of an allergic reaction. In the rare cases of reaction after injection of indocyanine green, these have been ascribed to the iodide contained in the molecule and only a few cases could be directly ascribed to the indocyanine molecule itself. The side effects reported comprise urticaria, nausea and anaphylactic choc with one reported case of death.7,8 In one reported study including 3774 ophthalmic patients 11 cases of minor reactions including 2 cases with an anaphylactic choc were reported.9 In a second study including 1226 patients 3 cases of nausea, 2 cases of urticaria, 2 cases of minor vaso-vagal choc and one case of severe choc were reported.10Although ICG is better tolerated than fluorescein, special care should be applied for patients known to be allergic to iodine. For these patients it is indispensable to use a iodide-free indocyanine green preparation produced in France under the commercial name of Infracyanine®.6

PHYSICOCHEMICAL PROPERTIES OF INDOCYANINE GREEN (ICG)

Infrared Fluorescence

The spectrum of maximal light absorption of the ICG molecule is situated around 800 nm and it emits in turn fluorescing light with a wavelenght of approximately 830 nm in the infrared spectrum of light.6 At these wavelengths the RPE is no more a significant screen

to infrared fluorescing light coming from the choroid, thus giving access to the choroidal vascular structures through the retinal pigment epithelium. As a consequence, it is important to realise that in ICGA the concept of masking effect does not play a determining role as it does in fluorescein angiography unless the masking elements are thick and/or heavily pigmented. Similarly the notion of window effect (window defet) that is so important for the interpretation of fluorescein angiography is not intervening significantly in the interpretation of ICG angiography as ICGA already “sees” through the retina and the pigment epithelium into the choroid. Therefore, ICGA is not useful in studying the pigment epithelium in contrast to fluorescein angiography which is the best modality to analyse RPE.

Macromolecular Behaviour

The molecular weight difference between ICG (775 daltons) and fluorescein (FNa / 354 daltons) molecules does not account for the specific distribution and behaviour obtained with ICG as compared to fluorescein. Beside the different wavelength at which ICGfluoresces,thecrucialdifferencebetweenthesetwo fluorescingmoleculescomesfromtheirbindingaffinity to proteins.6,11-13The ICG molecule is nearly completely protein bound and predominantly so to large sized proteins (lipoproteins),14 whereas only about 80% of the FNa molecules are protein bound and predominantly so to smaller proteins such as albumins.

At the level of the retina, the 20% free fluorescein leaks readily from slightly altered retinal vessels with minor damage to the blood-retinal barrier and readily impregnates tissues, whereas only major damage to retinal vessels allows ICG to leak15 (Figure 2A).

In the choroid, however, ICG leaks unimpaired but slowly from the fenestrated choriocapillaris16 (Figure 2B). During recirculation more and more ICG is entrapped in the choroidal tissue as the large ICGprotein complex is only slowly reabsorbed into the circulation and eliminated. Three days after intravenous ICG injection background fluorescence is still detected in the choroidal stroma. Fluorescein in contrast is quickly washed out from the choroid. Gradual ICG impregnation of the choroid occurs with time causing intermediate and late choroidal background fluorescence. This uniform choroidal impreg-

Figure 2A: Cartoon of slightly inflamed retinal vessel, showing that the small and non bound FNa molecule (yellow) egresses readily from the vessel, whereas the protein bound ICG molecule, formes a large complex and remains intravascularly

Figure 2B: Cartoon of choriocapillaris vessel, showing easy egression of both the FNa molecule and the ICG-protein complex

nation by ICG fluorescence is disturbed by choroidal inflammatory lesions such as granulomas, causing areas of decreased or absent fluorescence. It is this alteration of the slow physiological choroidal impregnation process that is the main parameter studied in ICGA performed for posterior uveitis. Indocyanine green choroidal impregnation is not only disturbed by space-occupying lesions (inflammatory foci) but also in case of inflammatory hypoperfusion or non-perfusion of the choriocapillaris that causes either complete or relative hypofluorescence in the area of the choriocapillaris where perfusion is disturbed. At the level of chorioretinal atrophic areas the macromolecular ICG-protein complex is incapable to

Figure 3: Comparison of FA and ICGA images of chorioretinal atrophy. FNa impregnates atrophic chorioretinal areas that appear hyperfluorescent on late frames (left frame), whereas these areas remain hypofluorescent on ICGA frames (right frame) (originally published and reproduced from: Ophthalmology 1998;105:432-40)

diffuse from adjacent areas and is not impregnating these atrophic areas that appear dark. In contrast, in fluorescein angiography, thanks to the small size of the fluorescein molecule the dye is diffusing towards atrophic areas that appear hyperfluorescent16 (Figure 3).

KEY POINTS: BASIC PRINCIPLES

•ICG is physiologically and massively extruding into the choroidal stroma from the largely fenestrated choriocapillaris but this low-grade background fluorescence was not detected earlier.

•ICG is much better tolerated than FNa but contains Iodide and iodide free ICG should be used in case of iodide allergy.

•ICG forms a macromolecular complex with large serum proteins that in physiologic conditions remains intravascularly, except at the level of the choriocapillaris where this macromolecular complex extrudes into the choroidal stroma where it is entrapped with a very slow wash-out.

•ICG also extrudes from inflamed large choroidal vessels and shows perivascular hyperfluorescence.

•ICG is fluorescing in the infrared spectrum that allows to get images through the RPE from inflammed choroidal vessels and inflammatory foci seen in negative because the ICG molecule is prevented from filling these space occupied by inflammatory foci.

ANGIOGRAPHIC TECHNIQUE (HARDWARE AND EXAMINATION TECHNIQUE)

ANGIOGRAPHIC SYSTEM

Two angiographic systems are principally used to perform indocyanine green angiography.17 The first system uses a conventional fundus camera linked to a digitisation system of images. This technique gathers the infrared fluorescence of all superimposed layers

of the fundus including retina and choroid. The advantage is not to miss lesions situated at different levels. Another advantage is the good visualisation of the periphery with good panoramic images as well as the good reproducibility of the angiographic exams which is very important for the follow-up of inflammatory patients. Finally this system gives good late phase images, which is another important point for inflammatory diseases.

The other system uses a scanning laser ophthalmoscope (SLO) which allows a confocal video-angio- graphy which allows to analyze the fluorescence in a very precise manner in one given layer. These systems are very useful when there is interest to study the fluorescence at a well determined level in the chorioretinal layers, such as for neo-vascular membranes or inflammatory diseases of the choriocapillaris for instance. In case of inflammatory diseases the lesions may be situated at different levels from the superficial level of the retina to the scleral level which may be more adequately imaged by the conventional fundus camera systems. Because the images are of such a precision in the focal plane with the systems using the SLO modality, it is more difficult to determine whether areas of hyperfluorescence or hypofluorescence are clinically relevant. Another disadvantage of the SLO systems is the lack of global imaging of the fundus although most of these systems provide now software programs that recompose a global image. Another possibility to have a global image is to use a Staurenghi contact lens that gives frames up to 140 degrees18 (Figure 4). Some of these systems provide also wideangle objectives that allow global fundus pictures. The late phase images can still be considered to be of inferior quality than the systems using conventional fundus cameras.

STANDARD ANGIOGRAPHIC PROTOCOL FOR INFLAMMATORY DISEASES OF THE FUNDUS

The information that is asked to ICG angiography in inflammatory eye diseases is different from what is needed for neovascular membranes. In the latter situation early frames and videoangiography are the most important angiographic features needed as we are looking for the feeder pattern of the neovascular membranes and the type and localisation of the membrane by analysing posterior pole hyperfluore-

Figure 4: Wide-angle fundus angiography, using the Staurenghi lens showing scattered stromal hypofluorescent dark dots in a case of sarcoidosis (Courtesy Alessandro Mantovani, Como, Italy

scence. In inflammatory eye diseases early frames are of less crucial importance.They are taken to determine whether there is a choroidal perfusion delay that can also be shown by fluorescein angiography. Most of the relevant information is coming from the later frames that show the physiologic egressing process of the ICG macromolecular complex from the choriocapillaris into the choroidal stroma completely filling the stroma with ICG molecules (Figures 5A and B). If there is choriocapillaris non perfusion or hypoperfusion, there is no such egress or diminished exudation and there will be relative or absolute hypofluorescence in the early and later angiographic frames (Figures 6A and B). Another situation that can impair the establishment of a normal diffuse choroidal background fluorescence is the presence of space occupying choroidal inflammatory lesions such as granulomas that will appear hypofluorescent against the fluorescing background because the ICG macromolecular complex cannot diffuse into that area (Figure 7A and B). Taking into account these particularities, a standard ICGA protocol to analyse choroiditis has been designed.16,19 The angioraphic procedure comprises an early phase up to 3 minutes, an intermediate phase (8-10 minutes) and a late phase (28-40 minutes). Before any dye injection pictures of both posterior poles are taken with the highest flash intensity to detect autofluorescence. Usually fluore-

Figure 5A: Cartoon of choroid showing outward diffusion of ICG from choriocapillaris into stroma (originally published and reproduced from: Ophthalmology 1998;105:432-40)

Figure 5B: ICG dye in stroma. Picture taken from an animation film showing impregnation of the stroma by the ICG-protein complex in the late phase of angiography when ICG has already been cleared from the general circulation making the large choroidal vessels appear dark (originally published and reproduced from: Ophthalmology 1998;105:432-40)

scein and indocyanine green angiography are performed in the same session. After the injection of 25-50 mg of indocyanine green (Cardiogreen®, Akorn Inc., Buffalo Grove Il, USA or Infracyanine®, SERB Laboratoires, Paris, France) diluted in 7.5 cc of a physiologic 0.9% saline solution, the early phase frames up to 2-3 minutes show superimposed retinal and large choroidal vessels and incipient exudation of the dye through the choriocapillaris into the choroidal stroma (Figure 8). At this point the angiogram can show retinal and choroidal perfusion delay that can also be shown by fluorescein angiography as choroidal fluorescein fluorescence is very intense in the first

Figure 6A: Choriocapillaris non-perfusion. Picture taken from an animation film showing dark areas at the level of the choriocapillaris in areas where there is choriocapillaris non perfusion

Figure 6B: ICGA frame of choriocapillaris non perfusion in APMPPE. Numerous geographic partly confluent hypofluorescent areas

seconds and can be seen through the pigment epithelium during the first 40 to 60 seconds of FA. The physiologic choroidal background fluorescence is analysed at 2 time points in the intermediate phase (8-10 minutes) and at the late phase (28 to 32 minutes or later in fairly pigmented patients) with pictures of the posterior pole as well as 8 panoramic pictures to visualise the fundus on 360°. The intermediate phase at about 8-10 minutes post-injection shows choroidal stromal background fluorescence and its anomalies such as hypofluorecent dark dots in case of stromal granulomas or geographic areas of hypofluorescence

Figure 7A: Stromal infiltration / mass due to inflammatory focus. Picture taken from an animation film showing absence of ICG impregnation in an area occupied by inflammatory iniltrate

Figure 7B: ICGA frame of stromal infiltrates. Numerous evenly distributed and regularly sized stromal foci in a case of Vogt-Koyanagi-Harada (VKH) disease

in case of choriocapillaris non perfusion and hyperfluorescence in case of leakage from the usually impermeable large choroidal vessels that appear fuzzy and indistinct in case of choroidal inflammation (Figure 9).

The late phase at about 28 minutes (or later in fairly pigmented patients) shows wash-out of the dye from the general circulation with the large choroidal vessels

Figure 8: Example of early phase ICGA frame. Irregular geographic hypofluorescent areas that result from choriocapillaris perfusion delay or non perfusion

Figure 9: Fuzzy indistinct choroidal vessels. Indistinct fuzzy choroidal vessels together with HDDs (top frame) the pattern of which is again well recognised after inflammation suppressive therapy (bottom frame)

Figure 10: Example of late ICGA frame. Large choroidal vessels appear hypofluorescent as ICG has been washed out from the general circulation

appearing dark against the background stromal fluorescence (Figure 10). If the hypofluorescent dark dots are still present in the late phase this means that the choroidal granulomas occupy the full thickness of the choroidal stroma.16 In case of choroidal inflammation this phase also shows either fuzzy vessels or diffuse choroidal hyperfluorescence produced by the abnormal exudation coming from large leaking normally impermeable choroidal vessels.

KEY POINTS: INDOCYANINE ANGIOGRAPHY: INDICATIONS, TECHNIQUE AND CHARACTERISTICS FOR INFLAMMATORY DISEASES OF THE FUNDUS

•If choroidal involvement is suspected dual fluorescein and ICG angiography is indicated (allows complete and thorough work-up of inflammatory involvement of all structures and is essential for follow-up of choroidal inflammatory lesions).

•Use a conventional fundus camera coupled to an image digitalising system (easier for peripheral imaging) or a scanning laser ophthalmoscopy system.

•Use one vial of 25 mg indocyanine green diluted in 7.5 cc of physiologic saline solution (Cardiogreen®, Akorn, Inc., Buffalo Grove, IL, USA). In case of iodine allergy use iodinefree Infracyanine® (SERB Laboratories, Paris, France).

•Angiographic procedure: (1) exclude autofluorescence by taking frames with the highest flash intensity previous to the dye injection; (2) perform early frames of the posterior pole up to 2-3 minutes (early phase); (3) perform posterior pole and 360° periphery panoramic frames at 8-12 minutes (intermediate phase); (4) perform fluorescein angiography between intermediate and late ICGA phases; (5) perform posterior pole and 360° periphery panoramic frames at 2835 minutes (late phase).

PRINCIPLES FOR THE INTERPRETATION OF

ICGA16,19

When analysing ICG angiography in posterior inflammatory disorders crucial differences with fluorescein angiography interpretation have to be born in mind to correctly analyse the images obtained.16,19 During initial circulation, ICG is comparable to fluorescein showing the passage through arteriovenous compartments except that it shows superimposed retinal and choroidal circulations. The difference occurs during recirculation time when ICG is progressively leaking out from the fenestrated choriocapillaris, gradually and physiologically impregnating the whole choroidal thickness (Figure 7A). In ICGA performed for inflammatory disorders, the main information is obtained less from the analysis of the early circulatory phase than from the analysis of the altered pattern of the filling of the choroidal space in the intermediate and late phases.

The physiologic filling process can be altered in 2 ways that can be associated in the same disease, either there is a decreased fluorescence or an increased fluorescence but usually both are associated.

ICG HYPOFLUORESCENCE (FLOW CHART 1)

The impregnation of the choroidal space by the ICG molecule can be absent or decreased (hypofluorescence) in at least two main situations.

1.There can be a decrease or absence of the physiological extrusion of the ICG macromolecular complex from the choriocapillaris (hypoperfusion or non perfusion). The areas that lack ICG or have a decreased ICG concentration due to decreased choriocapillaris perfusion appear hypofluorescent (Figures 6A and B). A good example to explain this process is the hypofluorescence seen after photodynamic therapy using Visudyne® that exactly shows hypofluorescence in the area where laser therapy was applied and where the choriocapillaris was damaged (Figure 11). In inflammatory diseases inflammation of the choriocapillaris leads to hypoperfusion and hypofluorescence. Because of irregular involvement of the choriocapillaris the hypofluorescent areas usually have an irregular geographic type of aspect (Figure 6B).

2.The second main mechanism at the origin of hypofluorescence is the impairment of the filling of the

Figure 11: Decrease of choriocapillaris exudation after photodynamic therapy. The diameter of the spot is easily identified by the round hypofluorescent area seen on ICGA

Figure 12A: Cartoon representation of whole thickness inflammatory lesion (originally published and reproduced from: Uveitis and Immunological Disorders. Edited by Pleyer U, Mondino B (Eds). Berlin, Heidelberg, New York: Springer; 2004)

choroidal tissue by the ICG molecule because of the presence of space-occupying lesions (inflammatory foci). All stromal inflammatory lesions (granuloma) are hypofluorescent (hypofluorescent dark dots, HDD) in the intermediate angiographic phase. If they remain hypofluorescent in the late phase this signifies that the inflammatory lesion occupies the whole thickness of the choroidal stroma (Figures 12A and B). This phenomenon is very well understood when looking at histological sections of choroidal stromal inflammatory diseases such as Vogt-Koyanagi-Harada (VKH) disease where the choroidal infiltrate can take the whole space from sclera to choriocapillaris (Figure 12C). When lesions

Figure 12B1: Full thickness inflammatory infiltrate. Hypofluorescent dark dots (HDD) are seen in the intermediate phase

Figure 12B2: Full thickness inflammatory infiltrate. Hypofluorescent dark dots (HDD) are present up to the late phase

become isofluorescent in the late angiographic phase, this means that inflammation causes only partial thickness infiltrates which delays fluorescence in these areas. In the adjacent areas fluorescence is already decreasing because of the washout of ICG, causing isofluorescence (Figures 13A and B).

Figure 12C: Histology showing full thickness granulomatous infiltration of the choroidal stroma in a case of Vogt-Koyanagi- Harada disease

Figure 13B2: Full thickness inflammatory infiltrate.

Hypofluorescent dark dots (HDD) are erased in the late phase

Figure 13C: Histology showing partial thickness granulomatous infiltration of the choroidal stroma in a case of Birdshot chorioretinopathy (Courtesy Prof Narsing Rao, Los Angeles)

Figure 13A: Cartoon representation of a partial-thickness inflammatory lesion (originally published and reproduced from: Uveitis and Immunological Disorders. Editedby Pleyer U, Mondino B (Eds). Berlin, Heidelberg, New York: Springer; 2004)

Figure 13B1: Partial thickness inflammatory infiltrate.

Hypofluorescent dark dots (HDD) are seen in the intermediate phase

Chorioretinal atrophy or choroidal stromal scarring will cause hypofluorescence from the early to the late phase of angiography, as the ICG-protein complex does not diffuse to these areas. These areas will remain hypofluorescent on follow-up angiograms.

Any thick or heavily pigmented mass in front of the choroid can be screen for ICG fluorescence and cause hypofluorescence from the early to the late phase of angiography by blocking fluorescence.

Another rare situation causing hypofluorescence that is not very well known is a pigment epithelium detachment under tension. Because the choriocapillaris is a low pressure capillary network only little pressure is needed to close down its aperture.

ICG HYPERFLUORESCENCE (FLOW CHART 2)

Impregnation of the choroidal space can be enhanced (hyperfluorescence) by increased leakage from the larger choroidal vessels that normally do not leak as explained by early publications on choroidal circulation using indocyanine green.2 We have plenty of histopathological evidence for choroidal vasculitis, either in VKH disease and more recently in a case of birdshot chorioretinopathy, clearly showing vasculitis of stromal vessels (Figure 14). In case of stromal

Figure 14: Birdshot chorioretinopathy. Histology of perivascular inflammation. (Courtesy Prof Narsing Rao, Los Angeles)

inflammatory vasculopathy, due to leakage, the vessels appear fuzzy in the intermediate angiographic phase (Figure 15A) and extrusion of the dye from large vessels causes late diffuse choroidal hyperfluorescence (Figure 15B).

In case of the presence of inflammatory foci in the choroidal stroma, diffuse hyperfluorescence is associated with hypofluorescent dark dots due to inflammatory infiltrates.

Hyperfluorescence occurs in at least four relevant situations.

1.As explained hereabove, in case of inflammatory choroidal vasculopathy additional leakage from normally impermeable choroidal vessels will add fluorescence to the physiological choroidal background fluorescence coming from the physiological choriocapillaris exudation. Because of this abnormal leakage choroidal vessels will appear

Figure 15A: Fuzzy indistinct vessels in the intermediate phase in a case of active birdshot chorioretinopathy. HDDs can be seen concomitantly

fuzzy in the intermediate and in the late phase. Instead of having a decrease of background fluorescence, there is a diffuse late phase hyperfluorescence due to leakage from larger non fenestrated choroidal vessels, a finding which will become obvious when looking at the posttreatment pictures showing well identifiable