Effects of epiretinal and subretinal delivery of ICG associated to light exposure during

vitreoretinal surgery in rabbits.

Mauricio Maia, MD*

Lukas Kellner MD*

Eyal Margalit, MD PhD*

Eugene de Juan, Jr, MD**

James Weiland, PhD**

Rhonda Grebe*

Gustavo Torres, MD*

Mark S. Humayun, MD, PhD**

*The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine,

Baltimore, Maryland, USA.

**The Retinal Institute at Doheny Eye Institute, The Keck School of Medicine at University

of Southern California , Los Angeles, California, USA.

Correspondence and reprint requests:

Mark Humayun

The Retinal Institute -Doheny Eye Institute

1450 San Pablo Street

Los Angeles CA 90033

humayun@usc.edu. Phone: 323-4426523/Fax: 323-4426519

Propriety interest: None

Key words: Indocyanine Green; iInternal lLimiting mMembrane; iIntraocular tToxicity; sSubretinal

dDelivery, phototoxicity, retina, vitreoretinal surgery.

ABSTRACT

Objective: to evaluate the effects of injected indocyanine green (ICG) onto the retinal surface

and into the subretinal space of rabbit eyes.

Methods: twenty–four (24) dutch-belted rabbits were studied. Four animals had optical nerve

head size measurement to standardize retinal light delivery and twenty animals were treated with

a two-port vitrectomy followed by ICGR 25 mg (Akorn – Buffalo Grove – IL) delivery ontoin the

retinal surface (8 animals) or into the subretinal space (12 animals). ICG was injected oninto the

retinal surface vitreous through a silicon soft-tipped cannula (Alcon - Forth Worth - TX). and a

A 43-Gauge Translocation Needle TM (Madlab- Baltimore –MD) was used to inject either create

subretinal blebs of BSSR (Alcon - Forth Worth - TX) or ICG in subretinal experiments. Light

was delivered over during 5 orand 7 minutes by connection of the endoilluminator to the

AccurusTM system (Alcon - Forth Worth - TX) or and MilleniumTM system (Bausch&Lomb/Storz-

Clearwater-FL). Animals were followed over various intervals the longest being 2 weeks (1 day,

7 days or 14 days) after surgical procedure. During follow up, fundus photography, fluorescein

angiography and indirect ophthalmoscopy were performed. Animals were sacrificed at different

time intervals (1 day, 7 days and 14 days after light delivery). Eyes were fixed and studied using

light microscopy.

Results: Minimal photoreceptors outer segments abnormalities were observed in animals that had

epiretinal ICG injections. Histological evaluation demonstrated that ICG injected into the

subretinal space (regardless of presence of light delivered via an endoilluminator) resulted in

damage of the outer segments. In some areas, both the photoreceptors and as well as the outer

nuclear layer were completely absent and RPE abnormalities (clumping, atrophy) were present.

These findings were more severe for longer intervals of follow up progressive over the duration of

the experiments. In the control eyes injected with BSS into the subretinal space, minimal outer

segments abnormalities were observed and the outer nuclear layer was normal over for the same

follow up period.

Conclusion: Subretinal delivery of 5mg/ml of ICG induces progressive RPE and photoreceptors

damage and results in a complete photoreceptor and outer nuclear layer degeneration 7 to 14 days

after injection. Thise kind of retinal overhelming damage was seen with due to subretinal ICG

injection regardless of endoiluminator use alone. Therefore, it was difficult to evaluate if precisely

additional damage if any was caused by a photodynamic effect due to endoillumination. Further

studies are necessary to elucidate these questions.

Cut this page

INTRODUCTION

Peeling of the Internal limiting membrane (ILM) and epiretinal membranes (ERM), are

commonly performed in vitreoretinal surgery 1-4 . These procedures may be difficult to carry out

due to poor visualization during surgery. Thus, techniques intended to that improve visualization

of these structures may facilitate the peeling procedure and reduce associated risks of retinal

damage.5

Indocyanine Green (ICG) is a tricarbocyanine dye that has the property to bind to

collagen fibrils type 4 and glicoproteins. During peeling procedure, it stains the internal limiting

membrane (and also other intraocular structures) and improves its visualization 5,6,7,8. However,

possibe ility of associated toxic effects haves been reported 9. It has been suggested that ICG

could induce or intermediate damage to the retinal pigment epithelium (RPE)9. The mechanisms

of these RPE abnormalities are not well understood and could be a result of several factors 9,10,11.

Since ICG absorbs the light wavelength between 600 to 900 nm, a photodynamic effect

was observed in tumors10 and keratocytes by using this drug intra venously (?) and irradiated the

lesion by infrared light11. In these experiments, the photochemical cellular injury may have been

caused by formation of singlet oxygen and lipid peroxides that resulted in a cellular rough

endoplasmic reticulum dilatation and chromatin condensation.11 We hypothesize the possibility

that, during macular hole surgery, dye could migrate into the subretinal space and cause a

chemical cellular injury associated or not with a photochemical effect secondary to the

endoilluminators light absorption by the ICG.

The objective of this study is to evaluate the effects of ICG in the epiretinal and

subretinal space of rabbit eyes and the role of the endoilluminator light as a possible ICG

photoactivator resulting in retinal damage.

We hypothesize that during macular hole surgery, dye could migrate into the subretinal

space and cause a chemical cellular injury with or without a photochemical effect secondary to

the endoilluminator light absorption by the ICG.

METHODS

INDOCYANINE GREEN (ICG) PREPARATION

1 vial of ICGR 25 mg (Akorn – Buffalo Grove – IL) was homogeneously diluted in 0.5 ml

of distilled water and 4.5 ml of BSSR, resulting in a 5mg/ml concentration and 270 mOsm.

Osmolarity was calculated measured (?) by a osmometer Osmomette ATM (Precision Systems Inc,

Sudbury, MA)

SURGICAL TECHNIQUE:

B1- OPTIC NERVE MEASUREMENTS

Four rabbits were sacrificied by overdosage of intravenous pentobarbital and the eyes were

removed. A sclerotomy was performed 2 mm posterior to the limbus, a 360 degrees sclerotomy

was completed and the anterior segment was removed. Vitreous was also removed and the optic

disk diameter was measured using a ruler (scale in microns) GIVE THE RESULTS OF THE

OPTIC NERVE MEASUREMENTS HERE. IT IS IMPORTANT FOR THE REST OF YOUR

METHODS.

A-IN VITRO EXPERIMENTS

In order to standardize the amount of light delivered into the retina, the endoilluminator

used during the “in vivo” experiments -( Model EL- (Synergetics-Saint Charles- MO) was fixed

in a plastic square in a horizontal position (Figure 1A). A 2.0mm diameter circle target

(according to our optic nerve measurements) was drawn in a white sheet of paper (target circle)

that was positioned vertically (in a orthogonal position) related to the endoilluminator (figure

1B). The endoilluminator was connected to the vitreous cutter machine AccurusTM (Alcon - Forth

Worth - TX) and or MilleniumTM (Bausch&Lomb/Storz-Clearwater-FL). The light source was

turned on and the plastic box and the endoilluminator were moved towards into the target

direction until the light source spot size was exactly the same size as the target diameter (figure

1A). The distance used in all surgical procedures between the tip of the light source and the

target was 2.5mm (see below) and this was the standard distance used in all surgical procedures

in rabbit eyes.

Light intensity was measured before each day of experiment by positioning the Photometer

probe IL1700 (International Light Inc- Newburyport- MA) 2.5mm far from the endoilluminator

(figure 1C). The light intensity was an average of all the measurements before each experiment .

B-IN VIVO EXPERIMENTS

Twenty –four (24) dutch-belted rabbits weighing 1.5 – 2.0 kg were used according to

Association of Research in Vision and Ophthalmology (ARVO) and the Johns Hopkins Hospital

guidelines for use of animals in experimental procedures. All surgical and clinical procedures

were performed under anesthesia using intra-muscular injection of 35 mg/kg of body weight of

ketamine hydrochloride (Phoenix Scientific Inc.- St Joseph- MO) and 5 mg/kg of body weight of

xylazine hydrocloride (Phoenix Scientific Inc - St Joseph - MO).

B1- OPTIC NERVE MEASUREMENTS

Four additional rabbits were sacrificied by overdosage of intravenous pentobarbital and

the eyes were removed. Esclerotomy was performed 2 mm posterior to the limbus, a 360 degrees

esclerotomy was completed and the anterior segment was removed. Vitreous was also removed

and the optic disk diameter was measured using especial ruler (scale in microns) XXZZZYYY..

B2 - SURGICAL TECHNIQUE OF ICG EPIRETINAL AND SUBRETINAL INJECTIONS:

Twenty rabbits were anesthetized as previously described and pupils were dilated with 2

drops of cyclopentolate hydrochloride 1% (Bausch&Lomb Pharmaceuticals Inc.- Tampa - FL)

and phenylephrine hydrochloride 5% (Bausch&Lomb Pharmaceuticals Inc.- Tampa - FL). A lid

speculum was placed and one drop of topical povidone-iodine 5% was instilled on the cornea.

After a few seconds the eye was washed using 10 ml of BSS.

Animals were divided in 2 groups:

I Group one – animals 1-8 (n=8) – ICG Epiretinal Experiments:

1 – Laser burns placement:

Laser burns were performed because …

Four laser burns were performed previously in one of the eyes of each rabbit using a

diode laser. (IRIDEX Inc –Mountain View – CA) Laser was set using the following parameters:

100 m diameter spot size, 100 mW power and duty cycle of 5% (what kind of laser-green red

yellow?).

Two laser burns were performed 4 mm inferior to the center of the optic nerve: One was

positioned 4 mm temporal lateral and the other 4mm nasal to the center of the optic disk. Two

additional laser burns were performed 4 mm superior to the center of the optic nerve: oOne of

the burns was placed 4 mm lateral temporal and other 4mm nasal to center of the optic disk

(figure 3A).

2 – Surgical technique

A hand-held contact lens for vitrectomy was positioned on the animal corneal surface.

The left eye was maintained in a position that exposed the optic disk right in the center of the

surgeon visual field by gentle eye traction using a 0.12 ocular forceps (company) in the limbus

superiorly exposed the optic disk right in the center of the surgeon visual field. The point used to

pull the eye in the limbus was marked. Two sutures of prolene 7-0 were placed either in the

temporal superior and nasal superior positions (4 to 6 mm far from the traction point) located

1mm posterior to the limbus. Two sclerotomies were performed using a 20-gauge microvitreal

retinal blade MVR (Alcon - Fort Worth - TX) in the superior nasal and superior temporal

positions 1 mm posterior to the limbus. A 2.5 mm infusion cannula was inserted in one of the

sclerotomies and stabilized with a 7-0 prolene suture (company).

2A – Light stimulation without ICG

These procedures were performed through either the nasal or the temporal sclerotomy,

depending on the infusion position. After the infusion was sutured, the endoilluminator was

inserted through the opposite sclerotomy. The endoilluminator distance from the retinal surface

was estimated by two parameters:

1- Size of the light spot : Position was adjusted until a round light spot of similar to the disk size

(around 1.5 to 2.0 mm) was seen achieved. The is distance between the endoiluminator and

the retina was estimated to be similar to the distance used in the “in vitro” experiments -

around 2.5mm (Figure 1).

2- Silicon-guided cannula: A silicon piece of 1.7 mm length was placed atin the end of the

endoilluminator. In all experiments and a light spot of 1 disk diameter size was shined

created inferior to the disk- before starting the light stimulation over the target area in all

experiments. In any of the experiments, Tthe end of the silicon piece guide was placed at a

long enough to touch the retina and we could observe a distance that ranged between1/4 to

1/3 of the silicon piece length from the between the end of the silicon tip and the retinal

surface, at an proving the estimated distance was of around 2.0 to 2.5mm.

The endoilluminator was held for 5 minutes in a position between one of the superior

lasers scars and the center of the optic nerve fiber layer. After 5 minutes of light exposure, the

endoilluminator was positioned in a similar distance from the retina during for 7 minutes in a

position between the inferior laser scar and the center of the opticnerve fiber layer.

The decision to use of testing 5 minutes and or 7 minutes of light exposure duration was

based in on pilot study results, in which 5 animals were exposed to MilleniumR light source

(Bausch & Lomb/ Storz – Clearwater – FL). Toxicity was not observed in after 5 minutes of

exposure holding the light source 2.,5 to 3 mm away far from the retina but it was present in after

10 minutes of light exposure holding the light source 2.5 mm far away from the retinal surface

using maximum light intensity and the an endoilluminator Model EL TM. (Synergetics-Saint

Charles- MO)

2B – Light stimulation with ICG – procedures performed inferiorly to the nerve fiber layer

A two-port vitrectomy was performed. The vitreous cutter tip was inserted and a

vitrectomy was completed with by removal of most of the care to remove the more quantity

possible of vitreous in contact to with the retinal surface. The vitreous cutter probe was removed

and a silicon soft-tipped cannula was inserted. A minimal amount of ICG was injected to verify

if the access of the drug had access to the retinal surface. If the drug could not reach the retinal

surface, the vitreous cutter tip was inserted again, vitrectomy was completed and 0.1 to 0.5 ml of

ICG 5 mg/ml was injected into onto the retina until the retinal surface turned into a green color.

A second infusion was placed in the opposite sclerotomy and the eye was temporarily

with 2 infusion lines to avoid hypotony. The first infusion was closed and removed from the eye

and the endoilluminator was inserted through this esclerotomy. Changing the infusion position

was important to allow the endoilluminator insertion in the same side of the retina that would be

exposed to light, resulting in a round spot configuration due to endoilluminator position

orthogonal to the target area, and also decreasing the possibility of touching the crystalline lens

due to endoilluminator manipulation inside the vitreous cavity.and held for 5 minutes in a similar

procedure described previously. At this time, Tthe endoilluminator was then held in a position

between one of the superior laser scars and the optic nerve head center fiber layer. After 5

minutes of light exposure, the position of the endoilluminator was changed between one of the

inferior laser scars and the optic nerve head center. Iit was similarly held 2 to 2.5 mm away far

from the retinal surface for during 7 minutes in this a position between one of the inferior laser

scar and the nerve fiber layer as described. Changing the infusion position was important to

allow the endoilluminator insertion in the same side of the retina that would be exposed to light,

improving the reproducibility of the light spot size (resulting in a round spot configuration due to

endoilluminator position orthogonal to the target area) and also decreasing the possibility of lens

touch due to endoilluminator manipulation inside the vitreous cavity. All surgical procedures

were performed with either both devices as the light source:

MillenniumR system (Bausch & Lomb- Chicago – IL) was used in 4 animals and AccurusR

system (Alcon - Dallas Fort Worth – TX) was used in other 4 animals. In all experiments, the

opposite eye was used as the control one (was any procedure done in the control eye?).

II - Group two – animals 10-20 (n=10) – ICG Subretinal Experiments

II.1 – Laser burns placement:

Procedure was similar to the one previously described in group I. Four laser burns were

placed using a diode laser (IRIDEX Inc - Mountain View - CA). Laser was set using the

following parameters: 100 m diameter spot size, 100 mwatts power and duty cycle of 5%:Two

laser scars were placed 4 mm inferior to the center of the optic nerve only: One of the scars was

positioned 4 mm lateral to the center of the optic nerve and other was positioned 4mm nasal to

the optic nerve (figure 3B). No laser was performed superiorly to the nerve fiber layer in these

subretinal experiments.

II.2 – Surgical technique

ICG and bBalanced Saline salt Solution solution (BSS) subretinal blebs placement

procedures associated withto light stimulation:

Two sutures were placed with vicryl 7-0 either in the temporal superior and nasal

superior positions followed by sclerotomies, infusion cannula was placed and Aa two-port pars

plana vitrectomy was performed as previously described. The vitreous cutter tip was inserted and

vitrectomy was completed with care to remove the more quantity possible of vitreous in contact

to the inferior retina. T After the vitreous cutter probe was removed, a 43-Gauge tTranslocation

nNeedle TM (Madlab- Baltimore –MD) was inserted through one of the sclerotomies and a

punctual retinotomy was created with the needle positioned right inferior to the laser scar (which

one- temporal or nasal?). ICG 5mg/ml was injected through the needle until and a bleb the size

of around 1-2 optic disks diameter was created (figure 4A). The same procedure was performed

above the superiorly to the laser scar (?) using the same needle. The 43-gauge needle was then

withdrawn and an endoilluminator probe was and held 2-2.53 mm away far from the retina for 7

minutes above in one of the ICGs blebs in an random alleatory manner. Care was taken to avoid

illumination over the adjacent ICG bleb that was separated from the previously illuminated bleb

by a laser scar. The endoilluminator was then withdrawn and the infusion was placed as

previously described. The a 43-Gauge tTranslocation nNeedle was inserted again through this

sclerotomy and to create another one bleb of bBalanced sSaline sSolution - BSS (Alcon - Forth

Worth - TX) was performed superiorly to the laser scar (I am not sure I understand where did

you place the ICG blebs and the BSS blebs, you have to use superior, inferior, nasal and

temporal in regard to the laser scars to make it clear) (figure 4B). After needle removal, the light

probe was inserted through the nasal sclerotomy and a light spot around 1.5-2.0-2.5 (you have to

be consistent) mm size was created superiorly to the laser scar (again, which scar?) that was

similar in size to the optic disk cup. After 7 minutes of light exposure, the vitrector tip was

inserted again and residual visible ICG was aspirated. The infusion was withdrawn and the

esclerotomies were closed. Right eyes served as were the control eyes ones (any procedure done

for them?).

LIGHT INTENSITY AND ESTIMATED IRRADIANCE:

The mean and standard deviation of light intensity was calculated based in on 6 different

measurements at in 6 different days for in each machine. The value was 4.26x106 ± 0.12 Candela

for the MillenniumTM system and 3.20x106 ± 0.09 Candela for the AccurusTM system.

FLUORESCEIN ANGIOGRAPHY (FA)

FA was performed by intravenous injection of 0.3 ml of sodium fluorescein (Alcon- Fort

Worth – TX) slowly (around 10-30 seconds) into the marginal ear vein. Fundus photographs

were taken using a fundus camera Topcon TRC (Topcon-Nagoya-Japan). Photographs were

taken a few seconds after injection and sequentially every 15-20 seconds for until 5 minutes had

been completed. Anterior chamber was observed for any leakage of sodium fluorescein. Eyes

that had evidence of diffuse fluorescence in the anterior chamber or abnormal leakage from the

retinal vessels were considered to have a blood-ocular barrier breakdown.

HISTOLOGICAL EVALUATION

Animals were sacrificed using by a 2ml intravenous pentobarbital injection. Eyes were

rapidly enucleated and fixed in a glutamate-formaline mixture for 24 hours (paraformaldehyde

2%, glutaraldehyde 2%, and phosphate buffer 0.1 M pH=7.4). Specimens were collected from

different areas in experimental eyes:

In eEpiretinal experiments (group XXX) had the samples were collected from in three

different serial sections performed 500m apart and starting 500m superior to from the upper

superior edges of the inferior (nasal/temporal ?) laser scar placed in the inferior retina. In

addition, three serial sections were also performed starting 500m inferior to the lower inferior

edges of the superior (temporal/nasal ?) scars placed in the superior retina (figure 3A).

In Ssubretinal experiments (group XXX) had the samples were also collected also in 3

different serial sections performed 500um apart and starting 500um superior and inferior from

the laser scar (again, specify). In these animals only two laser scars were placed in the inferior

retina (figure 3B). Samples were studied by light optical microscopy using toluidine blue ?

(company).

RESULTS

I – OPTIC DISK MEASUREMENT

Optic disk diameter in the 4 animals ranged as follows: 1.6mm in one animal 1.7 mm in

two animals and 1.8 mm in one animal. We considered 1.7 mm as a reference value (you should

put these results in the method section as mentioned previously. I would change the number you

use for the endoilluminator from 2-2.5 to 1.5-2.0 in every section you write about it (including

the methods)).

II - ANGIOGRAPHY

Fundus photography/fundus biomicroscopy Retinography of experimental group I eyes

(epiretinal injection of ICG) the epiretinal experiment one day after injection demonstrated

presence of laser scars and fundus photography/fundus biomicroscopyretinography of

experimental group II eyes (subretinal injection of ICG) the subretinal experiment in at the same

date time interval demonstrated 2 laser scars and 3 subretinal blebs (1 subretinal injection of BSS

and 2 injections of ICG - figure 5A). Fluorescein Aangiography (FA) demonstrated staining in

the areas all the extension of both the ICG and BSS subretinal blebs (figure 5B, 5C and 5D). A

point of leakage was observed in the area that The position that retina was perforated by the 43

gauge needle had a punctual leakage in all the blebs bubbles. In the ICG blebs that were exposed

irradiated by to light there was also a hypofluorescent ring near in the edges of the bleb

suggestive of a curtain effect due to blockage of the of a fluorescence blockage by the ICG

presence in the subretinal space. Surrounding this ring, there was also a hyperfluorescent ring in

the extreme periphery of the bleb. (figure 5B, 5C and 5D). Seven days after injection, minimal

residual ICG could be observed in a few animals (how many?) in the subretinal space (figure

6A). FA Angiography demonstrated multiple RPE changes abnormalities and presence of

window defects in positions of that ICG injections was injected into the subretinal space (figure

6B, 6C and 6D). No abnormalities were observed in blebs that BSS was injected. Similar

findings were observed 14 days after injection but RPE defects changes were more pronounced

prominent than one day and 7 days after ICG injection (figures 7A, 7B, 7C, 7D, 7E).

III - HISTOLOGY

IIIA- EPIRETINAL EXPERIMENTS (table 1)

No abnormalities were observed after ICG injection in onto the epiretinal surface and 5 or 7 minutes of

light exposure except from one animal that was exposed to light for 7 minutes that and had shortening

of the photoreceptors' outer segments (figure 8A).

IIIB-SUBRETINAL EXPERIMENTS (table 2)

Subretinal experiments demonstrated minimal/no outer segments abnormalities in animals

injected with subretinal BSS that BSS was injected into the subretinal space one day after

injection (figure 9A). AdditionallyHowever, when ICG was injected into the subretinal space,

there were abnormalities in of the photoreceptors outer segments and also pyknosis of the

photoreceptors outer segmentsnuclei were observed in the outer nuclear layer (figure 9B). These

abnormalities were also present when IGC was stimulated exposed to with light (figure 9C).

Seven days after injection, some residual BSS could be observed in the subretinal space and

minimal outer segments abnormalities were present (figure 10A). The outer nuclear layer and

RPE were normal. ICG injection into the subretinal space caused absence complete degeneration

of photoreceptors' outer segments, damage/pyknosis of the outer nuclear layer and RPE

abnormalities (figure 10B). These abnormalities were markedly presentmore prominent when

light exposure was added to subretinal ICG was exposed to light. The outer nuclear layer was

almost absent (figure 10C). Fourteen days after subretinal injection, the outer segments of

photoreceptors were minimally affected and no pyknosis could be observed in BSS subretinal

injection (figure 11A). In eyes that ICG was injected into the subretinal space, the dye could be

observed under the retina (figure 11B). No outer segments of photoreceptors could be observed,

RPE was completely severely damaged and pyknosis of the outer nuclear layer was present

(figures 11B and 11C), especially in the ICG group that where light was added (figure 11D).

Only one rabbit had a detectable lesion related to 5 minutes light exposure at 5 minutes

evaluation Histology also showeding outer segments shortening and misaligned associated to

with RPE damage (figure 9A) and this was interpreted as a phototoxic effect. (what about 7

minutes?

DISCUSSION (this section repeats the results too much)

The Iintraocular use of ICG enhances visualization of certain anatomical structures

during vitreoretinal procedures 5-7;8. However, there have been reports of RPE abnormalities and

poor visual acuity associated with it use 9. Many physiopathological mechanisms have been

postulated to explain these RPE abnormalities after ICG-guided peeling of the iInternal lLimiting

mMembrane:

1-Chemical toxicity affecting directly the retina TALK ABOUT ICG – IODATE RPE

RELATED PHOTOTOXICITY;

2-Phototoxicity associated with ICG absorption of the infrared spectrum of the endoilluminator

light resulting in an injurious photodynamic effect;

3 - Surgical trauma resulting in RPE abnormalities;

4 - Overexposure of to light during surgery. 12,13,14,15

In a previous study performed in our institution, 0.1 ml of ICG at a concentration of

5mg/ml injected and not removed from rabbit’s eyes demonstrated that this drug was related to

reversible changes in the electroretinogram ERG and also reversible ultra-structural cellular

abnormalities.

In the present study, no abnormalities or minimal photoreceptors outer segments

abnormalities had beenwere observed when ICG was injected epiretinally experiments were

performed using ICG confined to the epiretinal space and removed in at the end of the surgical

procedure (figure 8A).

FAngiography of performed one day after animals that subretinal injection of ICG was

injected under the retina and studied one day after surgical procedure demonstrated early staining

and progressive contrast leakage in positions areas that a bleb bubble was placed created (figures

5A, 5B and 5C). In addition, areas with fluorescence blockage could be also observed. The

Eedges of the subretinal ICG blebs that were exposed to light showed a leakage areasring. Seven

days after injection, angiography FA demonstrated RPE defects areas of hypo and

hyperfluorescence that were suggestive suggested of RPE damage and retinal atrophy (figure 6).

There were no more visible blebs XX days post injection (the retina was flat but residual ICG

could be also observed in the subretinal space of in XXsome animals). Edges of the subretinal

bleb exposed to light showed leakage areas (figure 7). Similar findings were present 2 weeks

after ICG subretinal injection (figure 8).

Histological evaluation 1 day after ICG subretinal injection demonstrated lesion

inshortening of the outer segments of the photoreceptors associated to with outer nuclear layer

damage (pyknosis) and RPE abnormalities (atrophy and proliferation) present in the ICG filled

bleb treated with ICG (figure 9B). The ICG bleb that was not exposed to light irradiated showed

similar findings (figure 9C). Seven days after injection, histology demonstrated absence of

retinal pigment epithelium, and photoreceptors outer segments and as well as nuclear pyknosis in

the outer nuclear layer with some areas of total absence of outer nuclear layer and

photoreceptors. consistent with apoptotic death (it could be consistent with many types of

damage and not only apoptotic death). These abnormalities were also observed 14 days after

injection. All these findings were observed in both areas – subretinal ICG injection + light and

also subretinal ICG injection only alone (figure 9).

Histological retinal evaluation 1 day after BSS injection demonstrated minimal

degeneration in the outer segments and no degeneration in the outer nuclear layer (figure 10A).

Similar findings were observed 7 days (figure XX) and 14 days (figure ZZ) after BSS subretinal

injection that areis consistent with the results of previous reports 17,18.

The presence of angiographic abnormalities and histologically abnormalities RPE and

photoreceptors damage in eyes injected with subretinal ICG associated compared to minimal

histological abnormalities in retinas exposed to light only with no ICG injection and or subretinal

injection of BSS allow us to conclude the that ICG by itself in the subretinal space of rabbits,

associated or not towith or without light exposure, result in damage to the outer retina and the

mechanisms of this damage deserves further evaluation.

Recently, Baumler and coworkers studied human skin and used ICG as a photodynamic

agent 11. In previous study, photoactivated ICG killed colonic cancer cells due to the generation

of singlet oxygen and subsequent formation of lipid peroxides, suggesting the basic mechanism

of ICG toxicity during photoactivation should be an oxidative process. 10, 11 No studies had been

reported regarding the injection of ICG into the subretinal space.

Berglin and coworkers determined, in New Zealand rabbits, that creating a stable

subretinal bleb of 1 to 1.5mm of 0.05 ml of subretinal sodium hyaluronate – Healon GV –

resulted in a degenerative process and decay of around 50% of the photoreceptors nuclei around

50% after 2 weeks post injection evaluation and around 40% in at the end of the first week17. In

our experiments, all the animals we could observe a complete degeneration in of the

photoreceptors' outer segments and nuclei and also the photoreceptors nucleuswas observed 1

week after injection. It has been reported that vitreous humor, aqueous humor and also serum

injected into the subretinal space of rabbits induced a retinal detachment that disappeared in 24

hours 18,19 Animals evaluated 1 week after detachment injection had a reattached the retina

apparently flat on clinical evaluation but in a fewXX (how many) animals, drug could be still be

observedalso visible in the subretinal space. The process of retinal degeneration after retinal

detachement appears to be a result of continuous elimination of photoreceptor cells and

apoptosis, but these abnormalities are more prominent 1 week or more of chronic detachment.17,

20, 21,22 It has been reported that vitreous humor, aqueous humor and also serum injected into the

subretinal space of rabbits induced a retinal detachment that disappeared in 24hours 18,19 In

addition, the process of retinal degeneration appears to be a result of continuous elimination of

photoreceptor cells and the apoptosis is probably the major contributor, but these abnormalities

are more important 1 week or more after chronic detachment.17, 20, 21,22

The following hypotheses try to explain the cellular damage observed in the subretinal

ICG blebs observed in our study:

1-There is drug degradation during over time, causing liberating liberation of sodium iodate 24

and this could result in RPE and retinal damage. XX

2-ICG could damage impair the RPE bump pump and therefore affect the diffusion of metabolic

substances from and to the choroid, impairing the convection of fluid and creating a barrier to

flow of fluid from the choroids and RPE to the neurosensory retina. In additionAs previously

mentioned, it is described that macromolecular high molecular weight drugs such as HealonR

could affect also the diffusion of substances from the retina to the choroids and decrease the Ph

of the subretinal space and also damage the outer retina. 17, 20

3 - ICG Bby dumping damaging the RPE, ICG could also contribute to persistent neurosensory

detachment resulting in retinal damage. or through a yet unknown process also directly damaging

the retina

4 – ICG could absorb the light and result in a photoxidative effect in the subretinal space as

discussed previously 10,11

We think tThis model is importantmight to generate questions about the effects of ICG in

rabbit eyes and try tohelp in understanding the effects of the ICG in human retinas. Further

studies are necessary to confirm and elucidate these hypotheses as well as to plan safe drug

deliveries and surgical techniques of ICG-guided peeling of the internal limiting membrane in

human vitreoretinal surgeries

CONCLUSIONS:

Subretinal delivery of 5mg/ml of ICG induces RPE and photoreceptors degeneration in

rabbits’ retina that is progressive during over time and results in a complete photoreceptor and

outer nuclear layer degeneration 7 and 14 days after ICG injection.

Mechanisms of RPE and photoreceptors injury as well as the role of light irradiation

exposure of the subretinal ICG must be better clarified. Further studies are necessary to elucidate

these questions.

REFERENCES

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Arch.Clin.Exp.Ophthalmol. 1999;237:648-53.

2. Mester V, Kuhn F. Internal limiting membrane removal in the management of full-thickness macular holes.

Am.J.Ophthalmol. 2000;129:769-77.

3. Park DW, Lee JH, Min WK. The use of internal limiting membrane maculorrhexis in treatment of idiopathic

macular holes. Korean J.Ophthalmol. 1998;12:92-97.

4. Lam DS, Chan WM, Tam BS. Macular hole surgery using thrombin-activated fibrinogen and selective removal of

the internal limiting membrane [letter; comment]. Retina 1999;19:478-79.

5. Kadonosono K, Itoh N, Uchio E, Nakamura S, Ohno S. Staining of Internal Limiting Membrane in Macular Hole

Surgery. Arch.Ophthalmol. 2000;118:1116-18.

6. Kadonosono K, Yazama F, Itoh N, Uchio E, Nakamura S, Akura J et al. Treatment of retinal detachment resulting

from myopic macular hole with internal limiting membrane removal. Am.J.Ophthalmol. 2001;131:203-07.

7. Kusaka S, Hayashi N, Ohji M, Hayashi A, Kamei M, Tano Y. Indocyanine green facilitates removal of epiretinal

and internal limiting membranes in myopic eyes with retinal detachment. Am.J.Ophthalmol. 2001;131:388-

90.

8. Tornambe, P. E. Intravitreal ICG dye enhaces vitrectomy surgery. http://www.vitreoussociety.org 12(1). 1999.

9. Pollack JS and Packo KH. Diffuse pigment epitheliopathy following intravitreal Injection of Indocyanine Green

(ICG). The Vitreous Society 18th annual meeting , 240. 1-8-2001.

10 Baumler W, Abels C, Karrer S, Weiss T, Messmann H, Landthaler M et al. Photo-oxidative killing of human

colonic cancer cells using indocyanine green and infrared light. Br.J.Cancer 1999;80:360-63.

11 Abels, C and Baumler, W. Indocyanine green and laser irradiation induce photoxidatiom. Arch.Dermatol Res

292, 404-411. 2001

12. Mester V, Kuhn F. Internal limiting membrane removal in the management of full-thickness macular holes.

Am.J.Ophthalmol. 2000;129:769-77.

13. Javid CG, Lou PL. Complications of macular hole surgery. Int.Ophthalmol.Clin. 2000;40:225-32.

14. Fuller D, Machemer R, Knighton RW. Retinal damage produced by intraocular fiber optic light.

Am.J.Ophthalmol. 1978;85:519-37.

15 Michels M. Intraoperative retinal phototoxicity. Int.Ophthalmol.Clin. 1995;35:157-72.

16. Marmor M. retinal detachment from hypeosmotic intravitreal injection. Invest Ophthalmol.Vis.Sci. 18(12),

1237- 1244. 2001.

17 Berglin L; Algvere P.V; Seregard S. Photoreceptor decay over time and apoptosis in experimental retinal

detachment. Graefe’s Arch. Clin. Exp. Ophthalmol. 1997; 235: 306-312

18 Imai, Z; Loewenstein, A; de Juan Eugene. Translocation of the Retina for Management of Subfoveal Choroidal

Neovascularization: Experimental Studies in Rabbit Eye. Am.J.Ophthalmol. 1998;125(5) 627-634.

19 Abe T; Yoneda S; Mori K; Hayashi N; Isono H. Reaction of the retina to injected subretinal fluids. J. Jpn.

Ophthalmol Sci 98: 453-462.

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humans. Arch. Ophthalmol. 1995 113: 880-886

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detachment. 1995 Invest. Ophthalmol. Vis. Sci. 36: 990-996

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Ophthalmol.Vis.Sci.1986 27:1770-1776.

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Table 1 - Summary of the most important histological findings in epiretinal experiments

Machine tested

Follow up

No Animals Light 5 minutes Light 7 minutes ICG 5 minutes ICG 7 minutes

Accurus

1 week

2 Normal Normal Normal Normal

Millenium

1 week

2 Normal Normal Normal Normal

Accurus

2 weeks*

2 Normal Normal *Abnormalities in

the OS; ONL and

RPE (2 animals).

*Abnormalities in

the OS, ONL and

RPE (1 animal).

Millenium \

2 weeks

2 Normal Normal Normal Normal

OS: outer segments; ONL: outer nuclear layer; RPE : retinal pigment epithelium

*Each of Tthese 2 animals had accidental migration of the drug accidentally into the subretinal space

during injection. In one of the animals the drug was observed in the subretinal space only in during the 5

minutes of light exposure.

Table 2 - Summary of the most important histological findings in subretinal experiments

Machine tested

Follow up

No Animals Light 7 minutes BSS ICG ICG + light 7

minutes

Accurus

1 week

2 Normal (2

animals)

Minimal damage

OS ( 2 animals)

Abnormalities in

the OS; ONL and

RPE (2 animals).

Abnormalities in

the OS; ONL and

RPE (2 animals).

Millenium

1 week

2 Normal (2

animals)

Minimal damage

OS ( 2 animals)

Abnormalities in

the OS; ONL and

RPE (2 animals).

Abnormalities in

the OS; ONL and

RPE (2 animals)..

Accurus

2 weeks

2 Minimal damage

of the OS

(1 animal)

Minimal damage

OS ( 2 animals)

Abnormalities in

the OS; ONL and

RPE (2 animals).

Abnormalities in

the OS; ONL and

RPE (2 animals).

Millenium \

2 weeks

2 Normal

(2 animals)

Minimal damage

OS ( 2 animals)

Abnormalities in

the OS; ONL and

RPE (2 animals).

Abnormalities in

the OS; ONL and

RPE . (2animals).

Accurus

1day

1 Normal Normal Abnormalities in

the OS; ONL and

RPE .

Abnormalities in

the OS, ONL and

RPE .

Milenium

1 day

1 Normal Normal Abnormalities in

the OS; ONL and

RPE .

Abnormalities in

the OS, ONL and

RPE .

OS: outer segments; ONL: outer nuclear layer; RPE : retinal pigment epithelium

LEGEND

Figure 1 – System used to standardize the distance between the top of the endoilluminator and

the retinal surface

A) A 2 mm diameter circle target was drawn in on a paper surface that was placed in an

orthogonal position related to the light source. The distance between the light source and the

target was estimated around to be 2.5 mm and this distance was used in the experimental

procedures.

B) The 2mm diameter circle visualized by on the back of the paper surface related in relation to the light

source (opposite surface from the light source).

C) The 2mm diameter circle by seen from a lateral view (same surface from the light source).

D) The photometer probe positioned 2.5 mm from the end of the endoilluminator.

Figure 2 – Measurements of the optic disk diameters:

A) A 360 degrees esclerotomy was completed and the anterior segment was removed.

B) The eyeball was opened and the optic nerve was measured using a especial ruler.

Figure 3 – Laser burns placement procedure before surgery:

A) Laser scars in epiretinal experiments.

Figure 4 – Surgical steps related to all procedures of the experiments

A) Vitrectomy before ICG injection

B) Cannula used for epiretinal experiments

C) Placement of two infusion cannulas to avoid hypotony during manipulation.

Figure 5 – Surgical steps of subretinal experiments

A) Laser scars in subretinal experiments.

B) Insertion of the 43-gauge cannula to create the subretinal blebs

C) Fundus photograph after ICG-bubble placementbleb procedure.

Figure 6 – Fundus photograph and FAangiography from subretinal experiments 1 day after

surgery:

A) Photograph from of a rabbit eye one day after ICG and BSS subretinal blebs placement

procedureof ICG and BSS. Note both ICG subretinal blebs (green blebs) adjacent to the left laser

scar. The inferior bleb – that was irradiated exposed with to light - hasand had an outer ring of

coloration slightly different from the retina. Note that this detail coloration is not present in the

superior ICG bleb. The BSS bleb is located superiorly to the right laser scar.

B) Early phase of a rabbit eye angiography FA from rabbit eye one day after ICG and BSS

subretinal blebs placement procedureof ICG and BSS. Note There is the diffuse staining of all

three blebs. There are also punctual leakage points areas (one in each bleb) that

representassociated with the 43-gauge needle penetration location trough the retinal surface.

Note in tThe inferior ICG bleb, which was exposed to(irradiated by light,) the presence ofhas a

hypofluorescent ring due to a “curtain effect”fluorescence blockage by the ICG from the ICG

dye blocking the fluorescence. This ring is surrounded by hyiperfluorescent circle.

B) Similar findings in thefrom an intermediate phase of FA (5 minutes)

C) Similar findings in thefrom a late phase of FA (10 minutes)

Figure 7 - Fundus photograph and angiography FA from taken during subretinal experiments 1

week after surgery:

A) Fundus photograph of a from rabbit eye 1 week after subretinal injection of ICG and BSS.

Note that both ICGs blebs are were positioned adjacent to the left laser scar. Residual ICG is still

visible in both ICG blebs and a line representing the previous BSS bleb is also visible near

around the right laser scar.

B) Early phase of FA angiography demonstrating staining of multiple RPE abnormalities -

staining -– under in both subretinal ICG blebs. Note the presence of hypofluoresent ring under in

both ICG blebs positions secondary to residual subretinal ICG 1 week after injection. The

inferior ICG bleb that was exposed irradiated to light for with 7 minutes of light (inferior) shows

a hyperfluorescent ring at the edge of the bleb. in the periphery This phenomena is not seen in

(that is not observed in other blebs). The retinal appearance above the BSS bleb (left bleb) is

normal and no abnormalities are evident in the position exposed to irradiated with light with no

subretinal injection only (inferior to the right laser scar).

C) Intermediate phase of FA angiography (5 minutes) demonstrating similar findings to of the

early phase. The hyperfluorescent ring around the subretinal ICG bleb that was exposed to light

is more intense than in the early phase (position that subretinal ICG was irradiated with light).

D) Late phase of FA angiography (10 ?5 minutes) demonstrating similar findings toof the

intermediate phase. The hyperfluorescent ring is even more intense than in the intermediate

phase (position that subretinal ICG was irradiated with light).

Figure 8 - Fundus photograph and FA angiography from subretinal experiments 2 weeks after

surgery:

A) Fundus photograph demonstrating the location of the previous two ICG blebs 2?1 weeks after

injection

B) Early phase of FA Angiography demonstrating mild RPE defects. The ICG bleb that was

exposed to light for irradiated with 7 minutes of light (right) shows hyperfluorescent ring in the

periphery (that is not observed in other blebs) – early phase.

C) Intermediate phase of FA Angiography demonstrating similar findings. (intermediate phase)

D) Late phase of FA Angiography demonstrating similar findings. (late phase)

E) Fundus photograph demonstrating 2 pigmented lesions representing the ICG subretinal blebs

(The lesions ar located at the left part of the figure lesions superior and inferior the to the left

laser scar). Superior to the right laser scar there is also a small area of mild retinal discoloration

color barely abnormal can be seen. This area representsing the previous BSS bleb (superior to

the left laser scar).

F) Late phase of FA (10 minutes) Angiography demonstrating the multiple RPE defects under in

both both ICG blebs that are located (inferior and superior to the left laser scar). Note the a

hyperfluorescent ring surrounding the RPE defects inferiorly to the left laser scar (late phase – 10

minutes). Also Nnote the mild RPE defects that are also present supreriorly to the left laser scar.

G) Fundus photograph demonstrating 2 pigmented lesions representing the ICG subretinal blebs

(left lesions around left laser scar). The inferior bleb – that was exposed to irradiated with light -

has an outer ring of coloration slightly different from the retinal color that is not present in the

superior ICG bleb.

Figure 9 – Histology specimen from an epiretinal experiments 24 hours after surgical procedure:

A) Outer segments abnormalities 1 day after 7 minutes of light exposure (toluidin blue x 400).

B) Histology specimen in which from accidentally ICG was delivered accidentally into the

subretinal space and exposed to light for associated to 5 minutes of light stimulation.

Photoreceptors and outer nuclear layer are completely severely damaged (only a few remaining

cells are visible). RPE cells are also damaged (toluidin blue x 400) I think you have to describe

more accurately what kind of damage.

Figure 10 – Histology from subretinal experiments 24 hours after surgical procedure:

A) Histology specimen from an area where BSS was injected into the subretinal space. The

Oouter segments of the photoreceptors are minimally shortened abnormal with the presence of

fluid edema between cells. The outer nuclear layer is normal (toluidin blue x 400).

B) Histology specimen from the site of penetration of the 43-gauge needle penetration through

the retina into the subretinal space. Note the abnormality in the retinal architecture due to needle

penetration. Outer segments of photoreceptors are slightly shortened abnormal but there is

pyknosis of the outer nuclear layer cells and an inflammatory reaction are also present in the

outer nuclear layer. There is also an inflammatory reaction due to trauma from the site of the 43-

gauge needle penetration into the subretinal space. Vacuolization of the RPE is visible as well

(toluidin blue x 200).

C) Histology specimen from a sight that was injected with subretinal ICG and exposed to light

for irradiated with light for 7 minutes in the subretinal space . This specimen is actually the in the

transition zone between normal and abnormal retinas. Outer Ssegments of photoreceptors are

misaligned and there is also abnormal amount fluid edema between cells and. There is also

ppyknosis of the cells in the outer nuclear layer . (toluidin blue x 200).

Figure 11 – Histology specimen from subretinal experiments 1 week after surgical procedure:

A) Histology taken from an area in which subretinal from BSS was injected.in the subretinal

space. Residual BSS is still present. Outer segments of photoreceptors are only slightly shortened

barely abnormal. There is a small amount of fluid also edema between photoreceptors. The outer

nuclear layer is normal. (toluidin blue x 400).

B) Histology specimen from an area injected with subretinal ICG in the subretinal space. Note

the cComplete degeneration of the photoreceptors outer segments and also pyknosis of the

outer nuclear layer can be seen. The RPE is also affected as well and vacuolization can be

observed ( toluidin blue x 400)

C) Histology specimen from an area injected with subretinal ICG and exposed to light for

Histology from ICG in the subretinal space irradiated with 7 minutes of light. Complete

degeneration of the photoreceptors outer segments and pyknosis of the outer nuclear layer can

be seen.Note the complete degeneration of the photoreceptors segments and also pyknosis of the

outer nuclear layer. RPE clumping and small areas of vacuolization can be observed (toluidin

blue x 400).

Figure 12 – Histology specimen from subretinal experiments 2 weeks after surgical procedure:

B) Histology from subretinal BSS injection site This is the actual location in the subretinal

space in the site of the 43-gauge needle penetration through the retina. Residual BSS is not

present. Outer segments of photoreceptors are slightly barely shortened and edematous abnormal

due to edema between photoreceptors and shortening. Vacuolization can also be observed. The

outer nuclear layer is normal. There is an mild inflammatory reaction (due to retinal penetration

with of the 43-gauge needle) (toluidin blue x 200).

B) Histology specimen from subretinal ICG injection site in the subretinal space. Note the

Ccomplete degeneration of the photoreceptors outer segments and pyknosis of the outer

nuclear layer can be seen.also pyknosis of the outer nuclear layer. The RPE is absent in some

areas. Note that ICG can be observed in the subretinal space and it is also being absorbed by

thein the choroids (toluidin blue x 400)

C) Histology specimen from a subretinal ICG injection site in the subretinal space. Note the

absence of the photoreceptors segments, diffuse RPE abnormalities -–clumping, - and also

vacuolization and . Ppyknosis of the outer nuclear layer. is also present. (toluidin blue x 400)

C) D) Histology specimen from a subretinal ICG injection site in the subretinal space

that was exposed to light for irradiated with 7 minutes of light. Note the

Ccomplete degeneration of the photoreceptors outer segments, diffuse RPE

abnormalities and also pyknosis of the outer nuclear layer can be seen. ( toluidin

blue x 400).

I am not sure how the figures look like but you might want to decrease the number of them. For

instance if figure 8 is almost like figure 7 you might want to show only part of figure 8 for new

findings etc.