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(Investigative Ophthalmology and Visual Science. 2007;48:1873-1883.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.06-0941
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In Vivo Retinal Tolerance of Various Heavy Silicone Oils

Jerzy Mackiewicz,1,2,3 Bastian Mühling,4 Wilfried Hiebl,4 Hasso Meinert,4 Kristel Maaijwee,1,5 Norbert Kociok,1,2 Christoph Lüke,1 Zbigniew Zagorski,3 Bernd Kirchhof,1,2 and Antonia M. Joussen1,2,6

1From the Department of Vitreoretinal Surgery, Center for Ophthalmology, and the 2Center for Molecular Medicine, University of Cologne, Cologne, Germany; the 3Department of Ophthalmology, Medical University of Lublin, Lublin, Poland; 4Fluoron GmbH, Neu-Ulm, Germany; 5The Rotterdam Eye Hospital, Rotterdam, The Netherlands; and the 6Department of Ophthalmology, University of Düsseldorf, Düsseldorf, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Heavy silicone oils are currently under investigation as a permanent tamponade in eyes with inferior PVR. This study was an investigation of Densiron 68 (Fluoron GmbH, Neu-Ulm, Germany) and several new heavy silicone oil admixtures on the basis of the perfluoroalkanes F4H5 (perfluorobutylpentane), F4H6 (perfluorobutylhexane), and F4H8 (perfluorobutyloctane) with respect to their long-term tolerance in a rabbit model.

METHODS. Because of the better solubility of the F4Hn-species (n = 5–8) in comparison to F6H8, we used F4H5, F4H6, and F4H8 to generate highly viscous, heavy silicone oils (HSO). After vitrectomy and fluid-air exchange, the left eye of each of five rabbits per group was filled with HSO 68-1500 (Densiron 68), HSO 45-5000, HSO 45-3000, HSO 46-5000, HSO 46-3000, HSO 48-5000, or HSO 48-3000, or pure F4H5, F4H6, or F4H8. Detailed clinical investigation, ERG testing, and histologic evaluation were performed throughout a 3-month follow-up.

RESULTS. Densiron 68 and HSOs based on F4H5, as well as the three control oils (silicone oil of 1000, 3000, and 5000 mPa · s) were well tolerated over 3 months. Histologically, the retina was unaffected. In contrast, intraocular inflammation, cataract formation, and retinal detachment and degeneration were noticed in all groups with HSOs based on F4H6 or F4H8.

CONCLUSIONS. Biocompatibility of the new HSOs is dependent on the lipophilic behavior (RF/RH ratio) and furthermore on the molecular dimension of the used semifluorinated alkanes (SFAs). HSOs on the basis of F4H5 may have advantages over silicone oils, on the basis of F6H8, for use as a tamponade agent for the inferior retina in difficult retinal situations.

Retinal detachment (RD) occurs in approximately 0.01% of the general population. In most cases, one intervention suffices to reattach the retina. However, in approximately 5% to 10% of RD, proliferative vitreoretinopathy (PVR) ensues1 and still is the most frequently occurring reason for surgical failure and blindness. PVR is a result of neuroectodermal or mesodermal contractile cells migrating in the vitreous and subretinal space stimulated by RD and breakdown of the blood-aqueous barrier.2 As a result, tractional cellular membranes develop, using vitreous collagen as scaffold. The result is tractional RD. The risk factors for PVR are trauma, RD, and endogenous factors, as well as repeated vitreoretinal surgery.3 The treatment of choice is vitrectomy, removal of periretinal membranes, and an endotamponade of gas or silicone oil. The role of the endotamponade is to seal retinal breaks, to confine eventual retinal redetachment to the tractional component,3 and possibly to displace the PVR-stimulating environment.

However, conventional silicone oil floats. Potential PVR-stimulating growth factors are concentrated in this subsilicone aqueous compartment.4 Therefore PVR RDs typically occur in the lower retina.5 PVR RDs require an endotamponade by a long-acting substance such as silicone oil.6 7 8 9 10

Perfluoroalkanes are heavier-than-water transparent liquids that are tolerated by the retina.11 Perfluorohexyloctane (F6H8) has recently gained attention as a long term vitreous substitute.12 However, surgeons are concerned about induced inflammation in the eye.13 14 15 Although F6H8 is chemically and biologically inert,11 its low viscosity promotes dispersion. Minute bubbles ("fish eggs") subsequently trigger chemotaxis of inflammatory cells and phagocytosis.16

Heavy silicone oil theoretically offers the chance to reduce the rate of tractional retinal redetachments17 through displacement of the PVR-stimulating environment from the inferior retina. Preliminary consecutive observations of 40 patients treated by a heavy silicone oil (HSO) tamponade18 and other series,19 20 do not disprove this hypothesis.

The HSO Densiron 68 (HSO 68-1500) is a transparent homogeneous liquid which is slightly heavier (1.06 g/cm3) than water and has a refractive index close to that of vitreous. Densiron 68 is a mixture of 5000 mPa · s silicone oil (specific gravity of 0.97 g/cm3) and of 3.5 mPa · s F6H8 (specific gravity 1.33 g/cm3). Densiron 68 has a low viscosity (1480 mPa · s) and interfacial tension (40.82 mN/m) rendering emulsification likely.

Clinically, silicone oils of higher viscosities have gained popularity due to the impression of surgeons that they are less prone to emulsification. Along these lines, it seems desirable to combine the advantages of high viscosity and increased gravity.

In this study, we investigated Densiron 68 and several new HSOs on the basis of perfluorobutylpentane (F4H5), perfluorobutylhexane (F4H6), and perfluorobutyloctane (F4H8) of variable viscosity and density, to determine their long-term tolerance in a rabbit model.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
A total of 75 eyes of chinchilla bastard rabbits weighing 2 to 3 kg were involved in the study. Animals were held in a 12-hour day-night facility in separate cages and fed regular chow. All experiments were approved by the local animal research review committee (Regierungspräsidium Köln) and performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. One eye only per animal was included, according to the ARVO guidelines.

Tamponading Agents
Each five rabbits received a 3-month tamponade with 1.5 mL silicone oil or balanced saline solution in the left eye. Details of the chemical and physical properties of the silicone oils used are listed in Table 1 .


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  		TABLE 1. Physical Properties of the Silicone Oils and SMAs Used

 
Because of the higher solubility of the F4Hn species in comparison to F6H8, we used F4H5, F4H6, and F4H8 to generate highly viscous HSOs. To achieve the high viscosity (3000 and 5000 mPa · s) and the required density (1.05–1.12 g/cm3), highly purified perfluorobutylpentane (F4H5), perfluorobutylhexane (F4H6), or perfluorobutyloctane (F4H8) were admixed with pharmaceutical grade silicone oils of 60,000 or 100,000 mPa. In particular, all HSOs of 5,000 mPa are mixtures of 60,000 mPa silicone oils and the respective semifluorinated alkane [SFA]. Conversely, the HSO of 3,000 mPa · s was generated of 100,000 mPa · s silicone oils and SFA.

As an exception, two silicone oils with 5,000 mPa · s viscosity were produced with F4H6, comprising either silicone oil of 60,000 mPa · s viscosity or 100,000 mPa.

The resultant oils were named according to their viscosity and the SFA used: HSO 45-5000, HSO 45-3000, HSO 46-5000 (HSO 46-5000 [Sil60T], and HSO 46-5000 [Sil100T]), HSO 46-3000, HSO 48-5000, and HSO 48-3000.

Silicone oils of 1000, 3000, and 5000 mPa served as controls, as did the pure SFA (F4Hn, n = 5, 6, or 8) and HSO 68-1500 (Densiron 68; Fluoron GmbH, Neu-Ulm, Germany), a mixture of 30.5 weight-% F6H8 and 69.5 weight-% silicone oil 5000. All tamponading agents were purified and bottled in sterile glass vials by the manufacturer (Fluoron). All silicone oils used were pharmaceutical grade oils.

After vitrectomy and fluid-air exchange, the left eye of each of five rabbits in each group was filled with one of the tamponading agents; one group with eyes filled with balanced saline solution served as the control.

Surgical Procedure
Before all surgical procedures, the rabbits were anesthetized by an intramuscular injection of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg). Pupils were dilated using 2.5% phenylephrine-0.5% tropicamide eye drops. Povidone-iodine 5% was applied to the eyelids. In addition, topical anesthesia was performed using several drops of lidocaine 0.4%. Two sclerotomies in the superior-temporal and superior-nasal quadrants (0.5–1.0 mm posterior to the corneoscleral limbus) were used for the insertion of a lighted infusion cannula and a pneumatic cutting device (OMNI, Ruck, Germany). Under constant infusion of a balanced saline solution, as much vitreous was removed as possible while avoiding lens damage. After fluid-air exchange, the vitreous cavity was filled with either of the tamponading agents or left with the saline (control group). The sclerotomies were closed with 7-0 Vicryl sutures. The operation was concluded by subconjunctival injection of gentamicin and betamethasone and application of gentamicin and atropine (1%) ointment.

Follow-Up Examination
During the observation period, the cornea, lens, vitreous cavity, and retina were examined daily for the first week after surgery, two times per week for the first month, and weekly up to 3 months. According to a prospective protocol, the following parameters were registered: corneal and lens opacities, the level and emulsification of the heavy liquid, formation of intravitreal strands, and visibility of the fundus. After surgery, a Schiötz tonometer, calibrated for the rabbit eye, was used to check intraocular pressure every day during the first week and weekly thereafter.

Electrophysiology
The electrophysiological testing was performed by one experienced examiner unaware of the appearance of the rabbit eye or type of vitreous replacement. To exclude the influence of potential circadian fluctuations all electroretinograms (ERG) recordings were performed in the morning at approximately 10 AM and recorded before surgery and 4 and 12 weeks thereafter. The scotopic triple-flash ERG was recorded together with the ISCEV (International Society for Clinical Electrophysiology of Vision) standard ERG responses. The animals were adapted to the dark for 30 minutes under general anesthesia. Pupils were dilated with 2.5% phenylephrine-0.5% tropicamide eye drops. Corneal contact lens electrodes (ERG-Jet; Universo Plastique SA, Le Cre-Du-Locle, Switzerland) were used for ERG recordings. A reference electrode was placed on the forehead. An ear-clip electrode served as the ground connection. The recordings were performed with a Ganzfeld system and computer-based signal acquisition and analysis software (Fa. Roland Consult, Wiesbaden, Germany).

Triple-Flash ERG
The scotopic triple-flash ERG was recorded after a dark-adaptation period of 30 minutes with fully dilated pupils. Three retinal light responses were elicited with flashes separated by short, dark intervals of increasing duration. The first flash was followed by a 140-ms dark period. Then a second flash was triggered. The third flash was triggered after a dark interval of 280 ms. The following cycle was started after a 560-ms interval, which resulted in a 980-ms stimulation cycle and a repetition rate of approximately 1 Hz. Averaging of 20 triple responses was performed for each flash luminance used. Different flash intensities of 0.01, 0.03, 0.1, and 0.2 cd/m2 were applied resulting in four averaged triple-response series.

For the triple responses, the b-wave amplitudes were measured, and the relative b-waves were calculated as the ratios of the b-waves at 140- or 280-ms intervals to the initial b-waves.

ISCEV Standard ERG
The ISCEV standard ERG was recorded. After the scotopic light response was recorded, the bright-flash response using the ISCEV standard flash of 2.4 cd-s/m2 was elicited. After light adaptation of 10 minutes with a steady background illumination of 10 cd/m2, photopic responses and the 30-Hz flicker ERG were recorded.

The a- and b-wave amplitudes and the implicit times of the standard responses were determined as well as the mean peak-to-peak amplitude of the photopic flicker ERG.

Histologic Examination
Eyes were enucleated 3 months after they were filled with the endotamponade. The 12 o’clock position at the limbus was marked by a suture, and eyes were immediately fixed in 4% paraformaldehyde for 24 hours. After tissue fixation, the globes were vertically sectioned, and the gross anatomy of the vitreous cavity and retina was observed.

Standard photographs in the middle periphery were taken of each animal’s upper and lower retina. After dehydration, the eyes were embedded in paraffin, processed for sectioning, and stained with hematoxylin and eosin (HE) and PAS (periodic acid-Schiff). Images of the gross anatomy and histology were captured with a charge-coupled device (CCD) camera (C4742-95; Hamamatsu Photonics, Hamamatsu City, Japan) attached to a microscope (Axioplane 2; Carl Zeiss Meditec GmbH, Oberkochen Germany). The images were viewed on a computer (G4; Apple Computer, Cupertino, CA) and analyzed (OpenLab software; Improvision Inc., Lexington, MA). The images were resolved at 1024 x 768 pixels and converted to tagged information file format (.tiff) files. Cell nuclei of the inner and outer nuclear layer were counted on standard magnification and expressed as cells per field. All quantification was performed in triplicate in a masked manner by a blinded observer.

Statistics
For analysis of the ERG responses, the Kruskal-Wallis test with a Mann-Whitney comparison. Results are presented as the mean ± SD. P < 0.05 was deemed significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinical Observation
There was no visible intraocular inflammation, or hyperemia of the retinal vessels observed after surgery in any of the control groups: silicone oil 1000, 3000, 5000, or Densiron 68. Indirect ophthalmic examination revealed a normal appearance of the retina, the retinal vessels, the optic nerve, and medullary ray throughout the observation period in these groups (Fig. 1A) . There was no difference among the study group and balanced saline-filled eyes.


Figure 1
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  		FIGURE 1. (A) Clinical appearance of the control groups: normal appearance of the retina and good fundus view in all groups. Of note, in the F6H8 group the meniscule is at the top, whereas it appears at the bottom in all silicone groups. (B) Clinical appearance of eyes with HSO 45-3000 or HSO 45-5000 tamponade. There is good visibility of the fundus and no macroscopic damage.

 
In none of the groups did we observe corneal neovascularization or clouding at any time during the follow-up, and there was no contact between the corneal endothelium and the tamponading agent in any of the animals. The intraocular pressure remained within the normal range (4–25 mm Hg) in all control groups during the follow-up period. The detailed results are shown in Table 2 .


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  		TABLE 2. Clinical Findings of Tamponade with Heavy Liquids and Controls

 
Only clinically significant cataract obscuring the fundus view was considered. The slight posterior cataract as seen in each two eyes of the silicone oil 3000 and the HSO 68-1500 group and in one eye of the silicone oil 5000 group did not intensify over time and was seen early on during the course, likely attributable to intraoperative lens damage.

Clinical examination of the HSO 45 groups demonstrated clinical tolerance equal to that in the controls. There was no inflammatory reaction visible. Cataract formation was seen only in two of five eyes with HSO 45-3000 and one in five eyes with HSO 45-5000. There was no fish egg formation with HSO 45-3000 or HSO 45-5000. There was no intravitreal inflammation, and the fundus was visible in all eyes filled either substance (Fig. 1B) .

In contrast, in groups after tamponade by HSO 46 or HSO 48, whitish precipitates on the surface of the HSO bubble were observed, starting 1 week after surgery. In the HSO 46 groups, similar precipitates were found on the posterior lens capsule. The more extended the inflammatory precipitates were 1 week after surgery, the more dense were the cataract formation and anterior synechiae 3 months after surgery.

Although not statistically significant, eyes filled with HSO 46-5000 produced from silicone oil of 60,000 mPa · s viscosity showed a more pronounced inflammatory reaction than did those filled with HSO 46-5000 produced from silicone oil of 100,000 mPa viscosity. In some animals, there was no view to the fundus in these eyes because of the dense cataracts. On macroscopic evaluation, the lens was completely disorganized in three of five eyes and seemed to be dissolved in the groups with the most severe reaction (Fig. 2A , Table 2 ).


Figure 2
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  		FIGURE 2. (A) Whereas no fundus view was possible in the groups HSO 46-3000, HSO 48-3000, and HSO 48-5000, there was a lesser reaction in the HSO 46-5000 group (admixture from 100,000 mPa viscosity). (B) A clear fundus view is present after 3 months of tamponade with F4H5, in contrast to the increasing cataract formation and obscured fundus view after F6H8 or F4H8 tamponade.

 
Nevertheless, HSO 46-3000, an admixture with Sil 100,000 mPa was less well tolerated than in both groups of HSO 46-5000. Similar to the findings just described, inflammatory reactions were visible, leading to cataract formation and intravitreal precipitation of cellular material with funnel RD.

The investigation of HSO 48-3000 and HSO 48-5000, demonstrated results comparable to those in the groups treated with F4H6. The fundus was obscured in all eyes at the end of the observation period, with changes similar to the gross pathology described earlier in two of three eyes.

F4H5 or F6H8 as a solvent did not result in an inflammatory reaction, whereas F4H6 and F4H8 led to an inflammatory reaction similar to that of the solvent-silicone oil admixtures. The anterior chamber in these eyes was shallow, sometimes completely flat; the whole eye was filled with a whitish material; and the retina was detached. Compared with the HSOs based on F4H6, the inflammatory state seemed to be pronounced and was prominent in all eyes. Similar findings were seen in all three eyes without exception, when pure F4H8 tamponade was tested. In general, in eyes with pure F4H6 and F4H8, disease was much more prominent if compared with HSO admixtures with the respective SMAs (Fig. 2B) .

Effect of Vitreous Tamponade on Retinal Function as Determined by ERG
Preoperative b-wave amplitudes for ISCEV standard-light stimuli ranged from 262.25 ± 86.78 to 520.50 ± 194.74 µV. There was no significant change in either the b-wave amplitude or latency in the control groups (Sil 1000, Sil 3000, Sil 5000, HSO 68-1500, or balanced saline surgery) during the course of the study (Table 3 , Fig. 3 ).


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  		TABLE 3. ERG Results

 

Figure 3
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  		FIGURE 3. Representative ERGs of heavy silicone groups before and after a 3-month tamponade.

 
There was a trend toward increasing b-wave amplitudes in eyes with a tamponading agent compared with the preoperative value. The alterations were predominantly found in b-wave amplitudes but were diminished in a-wave amplitudes. However, statistical analysis revealed no significance (P > 0.05) between preoperative b-wave amplitudes those measured 4 and 12 weeks after surgery. The amplitudes of flicker ERG demonstrated similar results.

In all HSO groups presenting without a clinical visible reaction, both scotopic and photoptic ERGs demonstrated similar variations for the experimental groups and controls (Table 3) . There was no significant change in b-wave amplitudes and latency during the follow-up of animals receiving tamponade with HSO 45-3000 or HSO 45-5000 or pure F4H5.

Using silicone oil admixtures with F4H6, similarly no significant changes were noted, however, no measurements were possible in selected animals with cataract and vitreous inflammation. For the same reason, no measurements were taken in the pure F4H6 group. For HSO 48-5000 there is a nonsignificant decrease of the b-wave amplitude during the course of 3 months (Fig. 3) . All other measurements in groups with admixtures with F4H8 did not show significant alterations.

Histologic Examination
Retinal layers were in a regular order after vitrectomy and light-microscopic examination after 3 months revealed no differences among the control groups with either BSS or silicone oil tamponade of either 1000, 3000, or 5000 mPa viscosity (Fig. 4A) . There were no nuclear drop-downs in the photoreceptor layer, degenerative changes in the ganglion cell layer, or visible fibrotic changes in the retina. Similarly, animals treated with HSO 68-1500 or F6H8 did not show evidence of structural damage on the light microscopic level. Preretinal macrophages and emulsified F6H8 bubbles were seen occasionally after tamponade with pure F6H8, as described earlier.21


Figure 4
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  		FIGURE 4. (A) Light microscopy of control groups: silicone oil 5000 mPa · s, HSO 68, and F6H8. In none of the groups was obvious structural damage observed. (B) Light microscopy of eyes filled with HSO 45-3000, HSO 46-3000, or HSO 48-3000. Of note, no visible damage was seen in the HSO 45-3000 group in contrast to both the other groups. The damage of the upper retina suggests rather toxic effects and not gravity-related damage. (C) Light microscopy of eyes filled with HSO 45-5000, HSO 46-5000, or HSO 48-5000. The most prominent changes were found in the group of eyes filled with HSO 48-5000, where no regular retinal structure was left. Magnification, x240.

 
Eyes filled with F4H5, HSO 45-3000, or HSO 45-5000 demonstrated a regular retinal structure. No obvious vacuolization, edema, or disarrangement of the layers was noted in any of the groups (Figs. 4B 4C 5) . Distinct areas demonstrated nuclear drop-downs. Rarely, a slight disarrangement of the outer nuclear layer was observed, but it could not be attributed to a single treatment group, as similar features were seen in selected cases in the control groups as well. Compared with the control groups, eyes with a heavy liquid endotamponade did not demonstrate a gross reduction in cellularity in the outer and inner nuclear layers.


Figure 5
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  		FIGURE 5. Histology and macroscopic appearance of eyes filled with either F4H5, F4H6, or F4H8. Although a regular layered structure is found in the group with F4H5 both in the upper and lower retina, the vitreous is completely filled with densely packed leukocytes, with a near complete destruction of the retinal layers both in the F4H6 and F4H8 groups. Details are shown together with the macroscopic appearance of the whitish material filling the vitreous cavity that interferes with the lens material.

 
With pure F4H5, single preretinal macrophages were observed in the lower part of the retina. All changes were classified as mild. In eyes with apparent emulsification, foam cells were seen on the lower retinal surface.

This result is in sharp contrast to the groups with F4H6 and F4H8 and the respective HSOs (Figs. 4B 4C 5) . The dense cataracts observed with the shallow anterior chamber were associated with a large PAS-positive thickening of the posterior capsule. In many cases the posterior capsule was damaged, and there was an invasion of leukocytes into the lens material. The vitreous body was, in many cases, completely filled with densely packed nucleated cells, mostly of the typical nuclear appearance of neutrophils (PMNs). There were no obvious foam cells as observed (e.g., in the F6H8-filled eyes). The retina in these severely damaged eyes was usually detached, and the layered structure was completely disorganized, indicating a long-standing retinal degenerative process. There were also some inflammatory cells within the degenerated retinal tissue. It appeared as if eyes in which the posterior lens capsule was damaged exhibited a larger inflammatory reaction compared with eyes with an intact posterior capsule and that the reaction between lens protein and the tamponading agents triggered the neutrophil reaction. The most severe inflammatory reactions with a nearly complete retinal degeneration were seen in the groups filled with pure F4H6 and F4H8, a less extensive reaction in admixtures of HSO and both substances. Similar to the clinical appearance, there was no correlation between the viscosity of the HSOs and the histologic appearance.

Even in eyes with a prominent inflammation of the posterior segment, there was no large inflammatory reaction in the anterior segment.

In eyes with less prominent inflammation (e.g., in the HSO 48-5000 group), degenerative changes both in the upper and lower retina were still detectable, shown as rarification of cells in both the inner and outer nuclear layers. Similarly, degenerative alterations were associated with nuclear drop-downs in the photoreceptor layer.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated in an animal model that a long-term tamponade with high viscosity HSOs based on F4H5 was tolerated. No retinal damage was detected with HSO 68-1500, HSO 45-3000, or HSO 45-5000.

However, silicone oils from admixtures with F4H6 and F4H8 demonstrated a limited intravitreal inflammatory reaction. This reaction was even more pronounced when pure F4H6 or F4H8 were used. In contrast, there was no comparable inflammation with pure F4H5 or F6H8. The vitreous cavity of eyes with F4H6 and F4H8 tamponade was filled with a whitish material consisting of densely packed granulocytes. The lens capsule was damaged and lens material and retinal tissue were infiltrated by inflammatory cells. The affection of the lens capsule and the immediate contact of inflammatory cells with lens material may have aggravated the pronounced reaction.

There are four mechanisms that could explain the phenomenon of the decreased tolerance: toxicity due to impurities, severe emulsification, gravity and mechanical damage, or direct toxicity of the substances.

Although previous reports have demonstrated a correlation between emulsification and volatile proportions rather than with the viscosity of the oils,22 there was no relation to the underlying silicone oil (silicone 60T or 100T mPa) in our current experiments. All silicone oils in these experiments used as pure substances or for the admixtures were highly purified and met the purity requirements for pharmaceutical-grade oils.

Furthermore, any contamination or impurities of the SMAs were excluded (F4H5, F4H6, and F4H8) by biocompatibility testing, including dermal irritation, dermal sensibilization, and cytotoxicity testing in cell cultures. Biocompatibility testing was performed according to international accredited procedures and recommendations (DIN [Deutsches Institut für Normung] EN [Europa Norm] ISO [International Standards Organization] 10993-10 (2002): "Tests for irritation and delayed-type hypersensitivity" as well as DIN EN ISO 10993-5 (1999): "Tests for in vitro cytotoxicity" (Test reports from BSL Bioservice, Planegg, Germany).

Third, the physical qualities of SMAs within the analyzed F4Hn series are relatively similar. Densiron (HSO 68-1500) has a specific gravity of 1.06 g/cm3, similar to HSO 46-5000 (1.064 g/ cm3) and HSO 48-5000 (1.054 g/ cm3), but the clinical and histologic appearance differs considerably. Similarly, we did not observe any difference both in the clinical and histologic appearance of the retina depending on the viscosity of the underlying silicone oils.

Thus, factors including density, viscosity, and surface tension cannot be relevant to the noted inflammation and to retinal intolerance.

We have previously demonstrated,11 21 that dispersion of F6H8 is irrelevant to retinal degeneration. However, it is well known that inflammatory reactions trigger emulsification and vice versa. Blood-ocular barrier breakdown as in uveitis and diabetes stimulates emulsification of silicone oil. Experimentally, the combination of tamponade vesicles with surfactants activates neutrophils or stimulates phagocytosis by monocytes.16 Still, in our experiments all animals were previously healthy and did not show any signs of underlying disease that increased the risk for inflammation. We observed a pure inflammatory reaction, but not an admixture of emulsification or fish egg formation known to occur from tamponades with SMAs.11 21

Thus, the intolerance demonstrated in this study seems to be more likely due to a direct reaction of F4H6 and F4H8 to the cells or cell membranes and the different retinal tolerance may be based on the chemical structure of the SMAs. The observed retinal tolerance in the group of the pure SMAs (F4Hn, with n = 5, 6, 8) correlates significantly with the length of the alkyl group. Whereas perfluorobutylpentane caused only a minimal and almost no inflammatory reaction in the rabbit eye, a much more pronounced reaction was seen after tamponade with perfluorobutylhexane and perfluorobutyloctane.

Chemically, SMAs are amphiphilic substances, whose lipophilic properties and thus the ability to penetrate cellular membranes are determined by the length of the alkyl-group.23 24 The lipophilic properties can be quantified by CTSH (critical temperature of solution in n-hexane).25 The CTSH is the temperature at which a defined mixture of substances does not homogenize. The smaller the CTSH value is, the more lipophilic the SMA. As cell membranes and other physiological borders are constituted of lipophilic substances, the CTS in olive oil is a good measure for the penetration characteristics into cells. Within the F4Hn group, there is a correlation between CTS and the clinical observation regarding the retinal tolerance.

There are also other measures of potential cell penetration and solubility of SMAs. According to Meinert and Roy,23 the chemical and also physical properties of SMAs are largely dependent on the length of the lipophilic alkyl chain (RH, NOH; number of hydrogen atoms) and the length of the lipophobic fluorinated carbon chain (RF, NOF, number of fluorine atoms). The longer the alkyl chain, the closer the properties to the corresponding hydrocarbon.23 24 Vice versa, a longer fluorinated carbon chain results in an SMA that is very close to the perfluorinated alkane. Thus, the proportion of the two chains can give an estimate of its solubility.

The ratio NOF/NOC and NOH/NOC compares the length of the fluoroalkyl chain (NOF) or the alkyl chain (NOH), respectively, to the total length of the molecule (NOC). Table 4 gives an overview on the relevant CTS values in olive oil, as well as the respective RF/RH, NOF/NOC, and NOH/NOC ratios.


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  		TABLE 4. CTS of Selected SFAs in Olive Oil and the Respective RF/RH, NOF/NOC, and NOH/NOC Ratios

 
Within one group (F4Hn and F6Hn) the CTS correlated with the RF/RH as well as the NOF/NOC and NOH/NOC ratios. In the F4Hn group the CTS decreased with increasing n. Thus, an increase in length by three CH2-groups from F4H5 to F4H8 resulted in a significant decrease of the CTS (by 70°C). Simultaneously, the RF/RH decreased from 0.80 to 0.50. This example demonstrates that small molecular changes (prolongation by three CH2 groups) caused a significant change in the physical and chemical properties and accordingly the differential biological tolerance to HSOs with admixtures of F4H6 and F4H8 compared with silicone oils with admixtures of F4H5.

When comparing CTS with the RF/RH of the respective group of SMAs in a linear regression, there was a significant linear correlation. The high values of the slope demonstrate the close association between the length of the alkyl group and solubility (Fig. 6) . In each group, a good correlation could be shown between the experimental results and the outlined theory (Table 4) 26 (Meinert et al. unpublished data, 2006).


Figure 6
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  		FIGURE 6. Correlation between RF/RH and CTS in the F4Hn and F6Hn groups.

 
As described earlier, the lipophilic behavior of the SMAs is explained by the length of the alkyl group. Thus, the RF/RH can be used to describe the penetration behavior. Within the F4Hn group, the tolerance falls in the range of around an RF/RH of approximately 0.7 to 0.8 (NOF/NOC value, 1.00–0.90). These estimates, however, are relevant for the F4Hn group only.

Similar to the pure F4Hn group, the retinal tolerance in the corresponding HSO group decreases with increasing n (n = 5, 6, or 8). Nevertheless, the observed reactions are much weaker than those seen with the pure substances.

Still, the reason for the extensive difference in retinal tolerance between F4H5 and F4H6 remains unclear. An increase in the methyl-group results in a measurable increase of the lipophilic properties (CST in olive oil decreases from 68–45°C), but this hardly explains the large differences in tolerance in the rabbit eye.

Similarly, a correlation of the known structural parameters like RF/RH, NOH/NOC, or NOF/NOC of one group of SMAs (e.g., F4Hn or F6Hn) does not result in a convincing correlation between structure and vitreal and retinal tolerability.

In conclusion, a relationship between the chemical structure of the respective SMA and its retinal tolerance is likely in this rabbit model, but not all adverse effects can be explained by the chemistry of the solvent. The experimental model itself may have an impact on retinal tolerance. It is well known, that rabbit eyes are very sensitive to surgical trauma and vitreous substitutes.21 The combination of a long-term tamponade and minimally increased lipophilic properties may lead to the difference in the retinal tolerance between F4H5 and F4H6.

Although not as prominent as in the rabbit model, there were inflammatory changes and cataract formation in clinical studies investigating HWS 46-3000 (consisting of 55 weight % perfluorobutylhexane and 45 weight% silicone oil 100,000 mPa · s) in 27 human eyes with a tamponade for 45 to 96 days.27 It is likely that the penetration abilities as seen with the pure substances are mitigated in the HSOs as the SMAs are embedded in silicone oil molecules.

Nevertheless, our data demonstrate that admixtures of F4H5 or F6H8 with silicone oil are safe to generate a HSO. Part of the intolerance of F4H6 and F4H8 and the respective silicone oils is explained by the chemical properties at the current state, however, we cannot draw final conclusions on the SMAs F4Hn and further studies are warranted.


   Acknowledgements
 
The authors gratefully acknowledge Doris Braune, Hanna Janicki, Martina Becker, Sabine Ricke, Frank Lacina, and Irene Söndtgen for technical assistance.


   Footnotes
 
Supported by the RetinoVit Foundation, Cologne, and a scholarship by KAAD (Katholischer Akademischer Ausländer Dienst) (JM). AMJ is a recipient of an Emmy Noether Grant (DFG Jo 324/6-2).

Submitted for publication August 9, 2006; accepted February 9, 2007.

Disclosure: J. Mackiewicz, Fluoron GmbH (F); B. Mühling, Fluoron GmbH (E); W. Hiebl, Fluoron GmbH (E); H. Meinert, Fluoron GmbH (E, P); K. Maaijwee, Fluoron GmbH (F); N. Kociok, Fluoron GmbH (F); C. Lüke, Fluoron GmbH (F); Z. Zagorski, Fluoron GmbH (F); B. Kirchhof, Fluoron GmbH (F); A.M. Joussen, Fluoron GmbH (F)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Antonia M. Joussen, Department of Ophthalmology, University of Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany; joussena{at}googlemail.com.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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