April 2011
Volume 52, Issue 5
Free
Cornea  |   April 2011
Corneal Endothelial Toxicity of Air and SF6
Author Affiliations & Notes
  • Hubert Landry
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
    Department of Ophthalmology, University of Montreal, Montreal, QC, Canada;
  • Anahid Aminian
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
    Department of Ophthalmology, University of Montreal, Montreal, QC, Canada;
  • Louis Hoffart
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
    Department of Ophthalmology, University of Montreal, Montreal, QC, Canada;
    Département d'ophtalmologie, Université de la Méditerranée, Marseille, France; and
  • Ossama Nada
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
  • Thouria Bensaoula
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
  • Stéphanie Proulx
    Laboratoire d'Organogénèse Expérimentale, Centre de recherche FRSQ du Centre hospitalier affilié universitaire de Québec, and Départements de chirurgie et d'ophtalmologie, Université Laval, Québec, QC, Canada.
  • Patrick Carrier
    Laboratoire d'Organogénèse Expérimentale, Centre de recherche FRSQ du Centre hospitalier affilié universitaire de Québec, and Départements de chirurgie et d'ophtalmologie, Université Laval, Québec, QC, Canada.
  • Lucie Germain
    Laboratoire d'Organogénèse Expérimentale, Centre de recherche FRSQ du Centre hospitalier affilié universitaire de Québec, and Départements de chirurgie et d'ophtalmologie, Université Laval, Québec, QC, Canada.
  • Isabelle Brunette
    From the Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada;
    Department of Ophthalmology, University of Montreal, Montreal, QC, Canada;
  • Corresponding author: Isabelle Brunette, Department of Ophthalmology, Maisonneuve-Rosemont Hospital, 5415, boulevard de L'Assomption, Montreal, QC, H1T 2M4 Canada; i.brunett@videotron.ca
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2279-2286. doi:10.1167/iovs.10-6187
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Hubert Landry, Anahid Aminian, Louis Hoffart, Ossama Nada, Thouria Bensaoula, Stéphanie Proulx, Patrick Carrier, Lucie Germain, Isabelle Brunette; Corneal Endothelial Toxicity of Air and SF6. Invest. Ophthalmol. Vis. Sci. 2011;52(5):2279-2286. doi: 10.1167/iovs.10-6187.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: The authors conducted in vivo assessment of corneal endothelial toxicity of air and SF6 in the feline model. This research was motivated by the increased use of air in anterior segment surgery in human subjects.

Methods.: This was a prospective masked study. The eyes of 16 healthy adult cats were randomly assigned for the injection of 0.7 mL air into the anterior chamber of one eye and SF6 in the contralateral eye. Daily examination included slit lamp photographs, pachymetry, and tonometry. Specular microscopy was performed before, 7 days after, and 10 days after injection. The animals were euthanatized, and the corneas were processed for alizarin red-trypan blue staining and for light and electron microscopy.

Results.: SF6 remained in the anterior chamber significantly longer than air. Both groups showed postinjection inflammation, which on average was maximal at day 2 and more severe with SF6. No difference in IOP was observed between the two groups. Specular microscopy showed significant endothelial cell loss in the SF6 group (mean postinjection cell loss, 132 ± 50 cells/mm2) but not in the group injected with air. Alizarin red staining revealed significant regional differences in cell density only in the SF6 group and more pronounced endothelial cell loss in the superior area.

Conclusions.: These results indicate that both air and SF6 injected into the anterior chamber of the eye can induce intraocular reaction in the feline model and that SF6 is more toxic than air in terms of endothelial cell loss and anterior chamber inflammation.

Injection of air into the anterior chamber of the eye has become standard practice in anterior segment surgery. The air bubble is used to exert direct pressure on an intraocular structure. Because air is less dense than aqueous humor, the bubble remains upward. The patient is asked to maintain the eye position that will optimize contact between the air bubble and the tissue layer to be stabilized. In Descemet stripping automated endothelial keratoplasty (DSAEK), 1 air is used to promote donor tissue adherence to the recipient bed, thus eliminating the need for sutures. The graft is centered over the posterior surface of the cornea, and the patient is asked to lie in a supine position and to look straight at the ceiling for 1 hour after surgery to distribute pressure from the air bubble over the graft surface. Similarly, the standard treatment for graft dislocation after DSAEK consists in air reinjection with head positioning. 2 Air can also be used to tamponade a detached Descemet's membrane after trauma or complicated cataract surgery. 3 For superior detachment, the patient is asked to keep his head in the upright position. Air has also been used recently to shorten the period of corneal edema when hydrops develops in keratoconus eyes. 4 Sulfur hexafluoride (SF6) has been proposed as an alternative to air because it remains in the anterior chamber for a longer period. 5,6 Although now routinely used in several anterior segment surgeries, the safety of air and SF6 for the corneal endothelium still must be demonstrated. The purpose of this study was to compare the in vivo corneal endothelial toxicity of air and SF6 in the feline model. 
Methods
All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the tenets of the Declaration of Helsinki. This research protocol was approved by the Maisonneuve-Rosemont Hospital Committee for Animal Protection (Montreal, QC, Canada). 
Population
Sixteen healthy adult cats (mean weight, 5.06 ± 0.44 kg; range, 2.2–8.6 kg) were obtained from a certified supplier. One eye of each animal received an intracameral injection of 20% (nonexpansive mix) 7 sulfur hexafluoride (SF6) (Labtician Ophthalmics Inc., Oakville, ON, Canada), and the other eye received a similar volume of filtered air. The choice of the eye was randomized, and all observers were masked for all steps (injection, daily examination, and endothelial cell morphometric analysis). 
Surgical Protocol and Medication
Surgery was performed under general anesthesia. Each animal was premedicated with an intramuscular injection of premixed acepromazine (0.05 mg/kg; Sanofi Aventis, Laval, QC, Canada), glycopyrrolate (0.01 mg/kg; Axcan Pharma, Mont Saint Hilaire, QC, Canada), and buprenorphine (0.01 mg/kg; Schering-Plough, Kirkland, QC, Canada) and was intubated. Anesthesia was induced and maintained by inhalation of isoflurane 2% (Baxter, Mississauga, ON, Canada). Atracurium (0.25 mg/kg, followed by 0.1 mg/kg every 20 to 30 minutes as needed; Sandoz, Boucherville, QC, Canada) was used to induce paralysis of the extraocular muscle. Full pupil dilation was obtained with topical administration of tropicamide 1% (Alcon, Mississauga, ON, Canada), phenylephrine 2.5% (Alcon), and cyclopentolate 1% (Alcon). Two 30-gauge needles were introduced through the limbus into the anterior chamber, and 0.7 mL air or SF6 was injected while the aqueous was tapped from the anterior chamber by passive filling of the second needle. The same procedure was performed in both eyes; the only difference was the nature of the injected gas. Wounds were checked for leaks. Betamethasone acetate and phosphate (3 mg in 0.5 mL; Sandoz, Boucherville, QC, Canada), tobramycin (10 mg in 0.25 mL; Alcon), and cefazolin (50 mg in 0.25 mL; Novopharm, Toronto, ON, Canada) were injected in the inferior fornix, and 1 drop of atropine 1% (Alcon) was instilled. Prednisone (5 mg/d; Apotex, Weston, ON, Canada) was given orally on a daily basis. 
Postinjection Follow-up
All animals were examined on a daily basis for 10 days after injection. Slit lamp assessment (Haag-Streit, Bern, Switzerland) was allowed to document the decrease in air and SF6 bubble size and the signs of inflammation. The size of the bubble in the anterior chamber was estimated by measuring the ratio between the diameter of the bubble and that of the corneal diameter in the vertical meridian. Aqueous flare and cell responses were graded according to the criteria of Schlaegel et al. 8 Intraocular pressure (IOP; Tonovet, TV01; Tiolat Oy, Helsinki, Finland) and central corneal thickness (CCT; Ultrasound Pachymeter SP 3000; Tomey, Nagoya, Japan) were measured on a daily basis. Superior corneal thickness (SCT) and inferior corneal thickness (ICT) measurements and central noncontact specular microscopy (Cellchek XL specular microscope; Konan Medical USA, Torrance, CA) were performed before surgery and 7 and 10 days after the injection. Specular microscopy photographs were taken in triplicate. Eleven animals were euthanatized (pentobarbital sodium 2 mL/4.5 kg IV; Sandoz) on day 10, three animals were euthanatized at 3 weeks and the two animals were euthanatized at 8 weeks. Both eyes were enucleated and examined. 
Tissue Preparation
Corneoscleral buttons were dissected and cut in three. A thin, vertical, central, 3-mm-wide band was fixed in glutaraldehyde 2.5% for transmission electron microscopy, and the nasal cornea was fixed in 10% formaldehyde for scanning electron microscopy. Specimens for electron microscopy were then processed as described in Proulx et al. 9  
The temporal cornea was used for vital staining. Two radial, 4-mm, full-thickness, peripheral relaxing incisions were made to allow flat mounting of the specimen. The endothelium was stained with trypan blue 0.25% and alizarin red S 0.2% (Sigma-Aldrich, Oakville, ON, Canada). 10 Three standardized photographs were obtained from each of the superior, central, and inferior endothelial areas (objective plan apo; 1.5×; SteREO Discovery V12; Carl Zeiss Canada, Toronto, ON, Canada). 
Endothelial Cell Morphometric Analysis
Endothelial cell densities and morphometric analyses of specular microscopy and stereo microscopy images were performed (KSS-409SP software version 2.10; Center Method) available on the Konan (Irvine, CA) noncontact specular microscope system. A minimum of 100 cells were counted in each studied area. The studied parameters included endothelial cell density (cells/mm2) and average endothelial cell area (μm2). The coefficient of variation (CV; SD of cell area/mean cell area) was used as a measure of polymegethism, and the percentage of hexagonal cells was used as an index of pleomorphism. 11,12 Bilateral endothelial morphometric analyses were performed. 
Analyses of the central corneal endothelium as a function of time (before and 7 and 10 days after injection) were based on in vivo specular microscopy. Because only central measurements can be taken with specular microscopy in the living animal, characterization of the geographic distribution of the endothelial cell damage across the corneal surface (superior, central, and inferior cornea) was made using vital staining of postmortem tissues. Alizarin red and trypan blue endothelial stainings were performed on all postmortem corneas. Enucleation was performed at 10 days in 11 animals, 3 weeks in 3 animals, and 8 weeks in 2 animals. Enucleation at day 10 was preferred to detect the early distribution pattern of endothelial damage before cell migration and uniformization of the endothelial mosaic. 13 15 The decision to postpone enucleation in five animals was based on the presence of a fibrin membrane attached to the corneal endothelium, either bilateral (four animals) or unilateral (in the SF6 eye of one animal). These membranes might have masked the endothelial mosaic and prevented postmortem morphometric analysis using a posterior approach. The five pairs of corneas with a longer follow-up were first compared with the 11 pairs with a 10-day follow-up. Because cell damage distribution was the same, all corneas were analyzed together. 
Statistical Analysis
We studied the effect of two factors (time and type of treatment) on the evolution of the measured parameters (e.g., bubble size, anterior chamber cells, flare). The two treatment modalities assessed were air and SF6. The effect of time was measured from day 0 to day 10 after injection. ANOVA with two repeated factors was used on full data sets. In case of an interaction between the type of treatment and time, each of these two factors was analyzed separately with repeated-measures ANOVA or paired t-tests while fixing the other factor. Mean and SEM are reported. Analyses were performed with SPSS (Chicago, IL) software, version 15.0, and P < 0.05 was considered statistically significant. 
Results
Bubble Size
Bubble size was studied as a function of time after injection and type of treatment. A significant statistical interaction was found between time and type of treatment (P < 0.001), and both parameters were found to affect bubble size. On the day of injection (day 0), the bubble size was the same in both groups (air and SF6), but it decreased significantly more quickly in the group injected with air (Figs. 1A–F, 2A). Complete clearance of the bubble in all eyes took 9 days with air (0.50% ± 0.35% of the anterior chamber volume at day 8 and no air at day 9) and >10 days with SF6 (2.19% ± 1.29% of the anterior chamber volume at day 10). 
Figure 1.
 
Slit lamp and vital staining assessment. (AF) In vivo slit lamp images of air (first column; right eye) and SF6 (second column; left eye) bubbles 1, 5, and 10 days (top, center, and bottom rows, respectively) after injection. The air bubble occupied 30% (A), 5% (B), and 0% (C) and the SF6 bubble occupied 50% (D), 35% (E), and 0% (F) of the anterior chamber 1, 5, and 10 days after injection, respectively. The fibrin membrane floating at day 5 in the anterior chamber of the eye injected with SF6 (white arrow) had retracted toward the angle superiorly at day 10. (GL) Ex vivo stereo microscopy images of the alizarin red and trypan blue-stained corneal endothelium of the same animal 10 days after the injection of air in the right eye (third column) and SF6 in the left eye (fourth column). The superior (first row), central (second row), and inferior (third row) corneal endothelial areas are shown (G, 2632 cells/mm2; H, 2688 cells/mm2; I, 2558 cells/mm2; J, 1527 cells/mm2; K, 2342 cells/mm2; L, 2717 cells/mm2). Scale bars: 100 μm (GL).
Figure 1.
 
Slit lamp and vital staining assessment. (AF) In vivo slit lamp images of air (first column; right eye) and SF6 (second column; left eye) bubbles 1, 5, and 10 days (top, center, and bottom rows, respectively) after injection. The air bubble occupied 30% (A), 5% (B), and 0% (C) and the SF6 bubble occupied 50% (D), 35% (E), and 0% (F) of the anterior chamber 1, 5, and 10 days after injection, respectively. The fibrin membrane floating at day 5 in the anterior chamber of the eye injected with SF6 (white arrow) had retracted toward the angle superiorly at day 10. (GL) Ex vivo stereo microscopy images of the alizarin red and trypan blue-stained corneal endothelium of the same animal 10 days after the injection of air in the right eye (third column) and SF6 in the left eye (fourth column). The superior (first row), central (second row), and inferior (third row) corneal endothelial areas are shown (G, 2632 cells/mm2; H, 2688 cells/mm2; I, 2558 cells/mm2; J, 1527 cells/mm2; K, 2342 cells/mm2; L, 2717 cells/mm2). Scale bars: 100 μm (GL).
Figure 2.
 
Slit lamp parameters, IOP, and central pachymetry values from day 0 to day 10. (A) Percentage of the anterior chamber occupied by the bubble. (B) Anterior chamber cells. (C) Anterior chamber flare. (D) Intraocular pressure. (E) Central corneal thickness. Slit lamp measurements were obtained daily on all eyes (n = 16 animals with complete data sets for air and SF6 from day 0 to day 10), whereas central pachymetry complete data sets could be obtained in only four animals (ultrasound pachymetry is not possible in the presence of a retrocorneal bubble). Error bars indicate SEM.
Figure 2.
 
Slit lamp parameters, IOP, and central pachymetry values from day 0 to day 10. (A) Percentage of the anterior chamber occupied by the bubble. (B) Anterior chamber cells. (C) Anterior chamber flare. (D) Intraocular pressure. (E) Central corneal thickness. Slit lamp measurements were obtained daily on all eyes (n = 16 animals with complete data sets for air and SF6 from day 0 to day 10), whereas central pachymetry complete data sets could be obtained in only four animals (ultrasound pachymetry is not possible in the presence of a retrocorneal bubble). Error bars indicate SEM.
Anterior Chamber Inflammation
A significant statistical interaction was found between time and type of treatment for each of the two parameters of anterior chamber inflammation (cells and flare; P < 0.001). Air and SF6 induced inflammation in both groups (Figs. 2B, 2C). This inflammation was significant from day 1 (P < 0.001 for cells and flare), reached a maximum on day 2, and decreased progressively. The amounts of cells and flare were greater and decreased significantly more slowly with SF6 than with air. Inflammation did not subside totally by day 10 (mean difference in cell grade between presurgery and day 10: air, 1.19 ± 0.23, P = 0.006; SF6, 1.69 ± 0.25, P < 0.001; mean difference in flare: air, 1.50 ± 0.30, P = 0.010; SF6, 2.25 ± 0.35, P = 0.001). In some cases, inflammation led to the formation of a loose fibrinous membrane in the anterior chamber, which tended to wrap the bubble and attach either to the iris surface or to the cornea. 
Intraocular Pressure
No statistical interaction was found between time and type of treatment. Intraocular pressure was affected by time after injection (P < 0.001) but not by type of treatment (air vs. SF6). In both groups, a temporary and nonsignificant increase in IOP was seen early after injection (mean increase, 11.4 ± 3.2 mm Hg; P = 0.255; Fig. 2D). IOP then decreased below preoperative levels (this decrease was significant from day 3) and remained low until the end of the 10-day study period. 
Corneal Thickness
Analysis of corneal thickness was limited by the high proportion of missing data because ultrasound pachymetry could not be performed in the presence of a retrocorneal bubble in direct contact with the cornea. Corneal thickness was measurable in all eyes before surgery. In eyes injected with air, time after injection and position on the cornea (SCT, CCT, ICT) had no significant effect on the ability to measure corneal thickness. In eyes injected with SF6, the percentage of available measurements was significantly affected by time and position on the cornea (on day 10, the percentages of available data were still inferior to normal: SCT, 25%; CCT, 56%; ICT, 94%; SCT, <ICT; P < 0.001). 
Central Corneal Thickness.
As a consequence, analyses on complete CCT data sets (air and SF6 from day 0 to day 10) could only be performed on four animals (Fig. 2E). No interaction was found between time and type of treatment. CCT was affected by type of treatment (central corneas were thicker with SF6 than with air; P = 0.019) but not by time of treatment. Corneas exposed to SF6 remained thicker until the end of the study period. 
Inferior and Superior Corneal Thickness.
Analyses on complete ICT data sets (air and SF6 from days 0, 7, and 10) could be performed on 13 animals. No significant interaction was found between time and type of treatment. The ICT was not affected by type of treatment, but it was affected by time after injection (P = 0.002). Paired comparisons revealed a significant postinjection increase in ICT (ICT: preinjection, 684 ± 18 μm; day 7, 732 ± 20 μm [P = 0.007]; day 10, 724 ± 21 μm [P = 0.046]). Statistical analyses could not be performed on SCT because of the high number of incomplete data sets. 
Specular Microscopy
Endothelial Cell Counts.
A significant interaction was found between time and type of treatment (P = 0.025). Although cell counts were similar in both groups before injection, they were significantly lower after injection in the SF6 group (mean air-SF6 difference before injection: −35 ± 37 cells/mm2, P = 0.357; after injection: 128 ± 41 cells/mm2, P = 0.008). Postinjection values consisted in the mean of day 7 and day 10 values. A mean cell loss of 132 ± 50 cells/mm2 was observed in the SF6 group (P = 0.021; Fig. 3A). 
Figure 3.
 
Preinjection and postinjection endothelial cell morphometric analysis of specular microscopy photos (n = 14 complete data sets). The mean of day 7 and day 10 values are reported for postinjection values. (A) Cell density. (B) Average cell area. (C) Coefficient of variation of cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 3.
 
Preinjection and postinjection endothelial cell morphometric analysis of specular microscopy photos (n = 14 complete data sets). The mean of day 7 and day 10 values are reported for postinjection values. (A) Cell density. (B) Average cell area. (C) Coefficient of variation of cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Average Cell Area.
Similarly, a significant interaction was found between time and type of treatment (P = 0.034). Although the average cell area was similar in both groups before injection, cells were significantly larger after injection in the SF6 group (mean air-SF6 difference: preinjection, 4.77 ± 6.12 μm2, P = 0.449; postinjection, −24.85 ± 8.72 μm2, P = 0.014; Fig. 3B). SF6 induced a significant increase in cell area, but air did not (mean increase: SF6, 25.52 ± 10.01 μm2, P = 0.024; air, −4.10 ± 5.56 μm2, P = 0.473). 
Coefficient of Variation of Cell Area.
No interaction was found between time and type of treatment. The CV increased after injection in both treatment groups (preinjection, 24.05 ± 0.68; postinjection, 28.58 ± 1.11; P < 0.001; Fig. 3C). The CV was also globally higher in the SF6 group (air, 25.86 ± 0.67; SF6, 28.03 ± 1.14; P = 0.007). However, because this difference was also present before injection, no conclusion could be drawn regarding the comparative effect of air and SF6 on the CV. 
Hexagonality.
Once again, no interaction was found between time and type of treatment. Hexagonality was affected only by time; both treatments induced a similar decrease in the percentage of hexagonal cells (before injection, 67.01% ± 1.06%; after injection, 61.87% ± 1.43%; P = 0.009; Fig. 3D). 
Alizarin Red and Trypan Blue Staining
Morphometric analyses demonstrated that signs of endothelial cell damage and instability were highly dependent on the position on the cornea (superior, central, or inferior), with a greater damage in the superior position. Figures 1G to 1L illustrate the typical aspect of the endothelium after injection of air in one eye (third column) and SF6 in the contralateral eye (fourth column). 
Endothelial Cell Density.
A significant interaction was found between position and type of treatment (P = 0.045). Although position on the cornea did not affect endothelial cell density in eyes injected with air, endothelial cell loss was significantly greater in the superior cornea of eyes injected with SF6 (mean difference in cell density with SF6: superior-center = −615 ± 186 cells/mm2, P = 0.016; superior-inferior = −1224 ± 290 cells/mm2, P = 0.003; center-inferior = −609 ± 189 cells/mm2, P = 0.018; Fig. 4A). In the superior position, however, the overall difference between the two groups only tended to be significant (mean air-SF6 difference: 619 ± 303 cells/mm2, P = 0.060). 
Figure 4.
 
Postmortem endothelial cell morphometric analysis after vital staining. Measurements were taken in three corneal positions (superior, central, and inferior) (n = 15 complete data sets). (A) Cell density. (B) Average endothelial cell area. (C) Coefficient of variation in cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 4.
 
Postmortem endothelial cell morphometric analysis after vital staining. Measurements were taken in three corneal positions (superior, central, and inferior) (n = 15 complete data sets). (A) Cell density. (B) Average endothelial cell area. (C) Coefficient of variation in cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Average Endothelial Cell Area.
No interaction was found between position and type of treatment, and neither position nor type of treatment was found to affect the average cell area (Fig. 4B). 
Coefficient of Variation of Cell Area.
No interaction was found between position and type of treatment. A significant position effect was observed (P = 0.011), but no differences were detected between air and SF6 groups (Fig. 4C). 
Hexagonality.
No interaction was found between position and type of treatment. The percentage of hexagonality was highly dependent on corneal position (P < 0.001). All paired differences were statistically significant (superior-center = −12.56% ± 3.78%, P = 0.015; superior-inferior = −20.99% ± 5.37%, P = 0.005; center-inferior = −8.43% ± 2.81%, P = 0.029). No differences were detected between air and SF6 groups (Fig. 4D). 
Scanning Electron Microscopy
Scanning electron microscopy was performed on four representative pairs of eyes. It confirmed the observations made by specular microscopy and vital staining, namely a greater endothelial cell damage in eyes exposed to SF6 than in those exposed to air, and a greater damage in the superior cornea, consisting of cell membrane disruption, missing cells, and increased endothelial cell area (Fig. 5A–D). Inflammatory cells scattered on the posterior surface of the cornea were occasionally found in eyes exposed to either air or SF6 (Fig. 5E). 
Figure 5.
 
Scanning electron microscopy. Two patterns of endothelial damage observed in the superior cornea of animals exposed to SF6 in one eye (left) and air in the other eye (right). Each row illustrates a pair of mate corneas. Damage was either similarly minimal (A, B) or significantly more important (C, D) in the eye injected with SF6 than in the mate eye injected with air. Endothelial cell damage included cell membrane disruption and missing cells. Patches of inflammatory cells scattered on the posterior corneal surface were occasionally seen (E). Scale bars: 50 μm (AD); 10 μm (E).
Figure 5.
 
Scanning electron microscopy. Two patterns of endothelial damage observed in the superior cornea of animals exposed to SF6 in one eye (left) and air in the other eye (right). Each row illustrates a pair of mate corneas. Damage was either similarly minimal (A, B) or significantly more important (C, D) in the eye injected with SF6 than in the mate eye injected with air. Endothelial cell damage included cell membrane disruption and missing cells. Patches of inflammatory cells scattered on the posterior corneal surface were occasionally seen (E). Scale bars: 50 μm (AD); 10 μm (E).
Transmission Electron Microscopy
In corneas exposed to SF6, it was found that the endothelial cells were thinner and more elongated than in the corneas exposed to air. Some endothelial cells appeared to be detached from Descemet's membrane and showed nonspecific protein accumulation in the subendothelial cell space. A definite disturbance in the structure of the mitochondrial cristae was observed and was a sign of cell stress. The inflammatory membrane seen over the endothelium included multiple cells, among which monocytes, immature fibroblasts, and epithelioid histiocytes (keratic precipitates) could be identified. Some similar but less advanced and more subtle changes were observed in the endothelial cells of corneas exposed to air. It could be seen that these cells were more viable. An example of the transmission electron microscopy aspect of the endothelium of a pair of corneas exposed to air and SF6 is shown in Figure 6
Figure 6.
 
Transmission electron microscopy. Pair of corneas of the same animal exposed to SF6 in one eye (A) and air in the other eye (B). In the cornea exposed to SF6, a nonspecific material lay in the subendothelial cell space. An inflammatory membrane, in which multiple cells such as monocytes, immature fibroblasts, and epithelioid histiocytes (keratic precipitates) were identified, covered the endothelium. No inflammatory membrane was found over the endothelial cells of cornea exposed to air. Scale bar: 2 μm (A, B).
Figure 6.
 
Transmission electron microscopy. Pair of corneas of the same animal exposed to SF6 in one eye (A) and air in the other eye (B). In the cornea exposed to SF6, a nonspecific material lay in the subendothelial cell space. An inflammatory membrane, in which multiple cells such as monocytes, immature fibroblasts, and epithelioid histiocytes (keratic precipitates) were identified, covered the endothelium. No inflammatory membrane was found over the endothelial cells of cornea exposed to air. Scale bar: 2 μm (A, B).
Discussion
Our results demonstrate that in the living feline model, both air and SF6 injected into the anterior chamber of the eye can induce an intraocular reaction. SF6 was more toxic than air in terms of anterior chamber inflammation (cell, flare, and corneal edema) and corneal endothelial cell losses, and the endothelial damage was significantly greater in the superior cornea. Air did not significantly affect central endothelial cell counts, and it had no effect on cell damage distribution. Both air and SF6 induced pleomorphism and polymegethism, two signs of reversible endothelial instability, which herein were primarily marked in the superior cornea. 
To the best of our knowledge, no previous report on the corneal endothelial toxicity of intracameral gases 16 21 compared air and SF6 toxicity in a living animal model with corneal endothelial wound healing characteristics similar to those of human subjects. The rabbit corneal endothelium model, which was used most often, 16 20 is known to differ significantly from the human model. Rabbit endothelial cells actively regenerate after wounding, showing extensive cellular division at the margin of the wound, migration, elongation, coalescence, endothelial multilayering, and replacement of old degenerated endothelial cells with new endothelial cells. 13,16,22,23 Endothelial repair in the rabbit is rapid and occurs in hours or days, depending on the severity of the damage. 13,23 Contrary to the rabbit, endothelium repair in the human takes place without mitosis. 24 26 Cells enlarge, spread, and migrate to cover the deficit left by the loss of neighboring cells, eventually restoring a more stable pattern but never returning to a normal size, and the cell count remains low. 27 The nonregenerative properties of the corneal endothelium of the cat were shown to be similar to those of the human corneal endothelium. 13 15,22  
Several older reports 16 21 using varying methodologies, mostly descriptive, occasionally quantitative, and usually in the rabbit model, all suggest some degree of corneal endothelial toxicity (corneal edema, aqueous flare and fibrin, and endothelial cell loss) for both air and expansive gases. Studies from Olson et al. 21 and Foulks et al. 17 report results in the cat model in accordance with our findings, showing signs of intraocular toxicity to air and SF6, respectively. 
As mentioned by Green et al. 18 and as suspected herein, longevity of the gas in the anterior chamber seems to play a key role in the severity of its deleterious effect. In the present study, the endothelial cell damage was located primarily in the superior cornea, where the gas bubble remained for the longest period. This finding parallels results from Doi et al. 28 showing increased retinal toxicity (abnormal glutamate distribution and thinning of the outer plexiform layer) of intravitreal injection of air, SF6, and C3F8 in the superior retina, where gases were in continuous contact. 
Endothelial toxicity may be explained by several mechanisms. Interference of the bubble with the aqueous humor nutrients 17,21 would most likely be limited to cases in which the bubble fills the entire anterior chamber, with smaller mobile bubbles allowing enough exchange between the endothelium and the aqueous humor. 
A mechanical interaction, resulting either from surface tension 29 or from direct trauma by the bubble itself, could be a possible source of endothelial cell damage. Hong et al. 30 used corneas mounted on an artificial anterior chamber filled at 40% with an air bubble and rotated 180° for 50 times to simulate the movement of an eye filled with air after DSAEK. The proportion of viable endothelial cells documented by trypan blue and alizarin red staining was significantly lower after air bubble trauma (79.8% ± 0.04%) than was the proportion of control fellow corneas (89.9% ± 0.02%; P = 0.03; n = 12 pairs). In the present study, the surgical stress was the same for all eyes and cannot be held responsible for differences between groups. 
Endothelial cell injury could also be secondary to inflammation, which in our study was more severe with SF6 than with air. An inflammatory reaction in cat and rabbit eyes after the intracameral injection of air, SF6, or C3F8 has been reported by others. 17,19 The inflammatory response observed herein in cat eyes was more marked than that routinely observed in human eyes, such as after DSAEK surgery. 
Changes in the antioxidant system of the aqueous humor have also been reported after intravitreal injection of SF6, as measured by the increased activity of catalase and superoxide dismutase and increased malondialdehyde concentrations. 31 These authors concluded that the increased occurrence of active oxygen species in the aqueous humor leads to insufficiency of the antioxidant system and intensification of the peroxidation processes, reflected by increased malondialdehyde concentration. 
It should be mentioned that the numerous reports on the beneficial effect of air injected into the anterior chamber to prevent its collapse during intracapsular or extracapsular cataract extraction 32 39 were of no help in the present study. In these older reports, the considerable mechanical protection offered by the short-term (few minutes only) filling of the anterior chamber with air would have masked any eventual sign of air endothelial toxicity. 
In conclusion, both air and SF6 injected into the anterior chamber of the eye were shown to induce intraocular reaction in the living feline model. SF6 was more toxic than air in terms of endothelial cell loss and anterior chamber inflammation. Based on the results obtained herein, the lack of endothelial toxicity of air and SF6 in the human subject should not be considered as granted. We therefore recommend that these gases be used with caution in the anterior chambers of human subjects. Their use should be at the minimal dose necessary to obtain the desired tamponade effect and for no longer than required, and air should be favored over SF6. 
Footnotes
 Supported by the Fonds de la recherche en ophtalmologie de l'Université de Montréal (FROUM), the FRSQ Research in Vision Network (IB, AA), and the Canadian Institutes of Health Research (CIHR IB, LG). LH held a postdoctoral scholarship from the Société Française d'Ophtalmologie, Paris, France. ON held a postdoctoral scholarship from the Egyptian Ministry of Higher Education, Egypt. IB holds the Charles-Albert Poissant Research Chair in Corneal Transplantation, University of Montreal, Montreal, Canada. LG holds the CIHR Canadian Research Chair in Stem Cells and Tissue Engineering.
Footnotes
 Disclosure: H. Landry, None; A. Aminian, None; L. Hoffart, None; O. Nada, None; T. Bensaoula, None; S. Proulx, None; P. Carrier, None; L. Germain, None; I. Brunette, None
The authors thank Michel Asselin, John Douglas Cameron, Michel Carrier, Miguel Chagnon, Marie-Eve Choronzey, André-François Couture, Marie-Josée Guyon, Julie Dubeau, Angèle Halley, Serge Rosolen, Denis Sherknies, and Marian-Ananda Zaharia for their technical assistance and professional advice. 
References
Gorovoy MS . Descemet-stripping automated endothelial keratoplasty. Cornea. 2006;25(8):886–889. [CrossRef] [PubMed]
Price FWJr Price MO . Descemet's stripping with endothelial keratoplasty in 200 eyes: early challenges and techniques to enhance donor adherence. J Cataract Refract Surg. 2006;32(3):411–418. [CrossRef] [PubMed]
Mahmood MA Teichmann KD Tomey KF al-Rashed D . Detachment of Descemet's membrane. J Cataract Refract Surg. 1998;24(6):827–833. [CrossRef] [PubMed]
Miyata K Tsuji H Tanabe T Mimura Y Amano S Oshika T . Intracameral air injection for acute hydrops in keratoconus. Am J Ophthalmol. 2002;133(6):750–752. [CrossRef] [PubMed]
Mearza AA Qureshi MA Rostron CK . Experience and 12-month results of Descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea. 2007;26(3):279–283. [CrossRef] [PubMed]
Zusman NB Waring GO3rd Najarian LV Wilson LA . Sulfur hexafluoride gas in the repair of intractable Descemet's membrane detachment. Am J Ophthalmol. 1987;104(6):660–662. [CrossRef] [PubMed]
Abrams GW Edelhauser HF Aaberg TM Hamilton LH . Dynamics of intravitreal sulfur hexafluoride gas. Invest Ophthalmol. 1974;13(11):863–868. [PubMed]
Schlaegel TFJr O'Connor GR . Current aspects of uveitis: general considerations. Int Ophthalmol Clin. 1977;17(3):1–42. [CrossRef] [PubMed]
Proulx S Audet C Uwamaliya J . Tissue engineering of feline corneal endothelium using a devitalized human cornea as carrier. Tissue Eng Part A. 2009;15(7):1709–1718. [CrossRef] [PubMed]
Taylor MJ Hunt CJ . Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction. Br J Ophthalmol. 1981;65(12):815–819. [CrossRef] [PubMed]
Bergmanson JP . Histopathological analysis of corneal endothelial polymegethism. Cornea. 1992;11(2):133–142. [CrossRef] [PubMed]
McCarey BE Edelhauser HF Lynn MJ . Review of corneal endothelial specular microscopy for FDA clinical trials of refractive procedures, surgical devices, and new intraocular drugs and solutions. Cornea. 2008;27(1):1–16. [CrossRef] [PubMed]
Van Horn DL Sendele DD Seideman S Buco PJ . Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci. 1977;16(7):597–613. [PubMed]
Ling TL Vannas A Holden BA . Long-term changes in corneal endothelial morphology following wounding in the cat. Invest Ophthalmol Vis Sci. 1988;29(9):1407–1412. [PubMed]
Huang PT Nelson LR Bourne WM . The morphology and function of healing cat corneal endothelium. Invest Ophthalmol Vis Sci. 1989;30(8):1794–1801. [PubMed]
Van Horn DL Edelhauser HF Aaberg TM Pederson HJ . In vivo effects of air and sulfur hexafluoride gas on rabbit corneal endothelium. Invest Ophthalmol. 1972;11(12):1028–1036. [PubMed]
Foulks GN de Juan E Hatchell DL McAdoo T Hardin J . The effect of perfluoropropane on the cornea in rabbits and cats. Arch Ophthalmol. 1987;105(2):256–259. [CrossRef] [PubMed]
Green K Cheeks L Stewart DA Norman BC . Intraocular gas effects on corneal endothelial permeability. Lens Eye Toxic Res. 1992;9(2):85–91. [PubMed]
Lee DA Wilson MR Yoshizumi MO Hall M . The ocular effects of gases when injected into the anterior chamber of rabbit eyes. Arch Ophthalmol. 1991;109(4):571–575. [CrossRef] [PubMed]
Leibowitz HM Laing RA Sandstrom M . Corneal endothelium. Arch Ophthalmol. 1974;92(3):227–230. [CrossRef] [PubMed]
Olson RJ . Air and the corneal endothelium: an in vivo specular microscopy study in cats. Arch Ophthalmol. 1980;98(7):1283–1284. [CrossRef] [PubMed]
Bahn CF Glassman RM MacCallum DK . Postnatal development of corneal endothelium. Invest Ophthalmol Vis Sci. 1986;27(1):44–51. [PubMed]
Fukami H Laing RA Tsubota K Chiba K Oak SS . Corneal endothelial changes following minor trauma. Invest Ophthalmol Vis Sci. 1988;29(11):1677–1682. [PubMed]
Doughman DJ Van Horn D Rodman WP Byrnes P Lindstrom RL . Human corneal endothelial layer repair during organ culture. Arch Ophthalmol. 1976;94(10):1791–1796. [CrossRef] [PubMed]
Kaufman HE Capella JA Robbins JE . The human corneal endothelium. Am J Ophthalmol. 1966;61(5 pt 1):835–841. [CrossRef] [PubMed]
Matsuda M Suda T Manabe R . Serial alterations in endothelial cell shape and pattern after intraocular surgery. Am J Ophthalmol. 1984;98(3):313–319. [CrossRef] [PubMed]
Waring GO3rd Bourne WM Edelhauser HF Kenyon KR . The corneal endothelium: normal and pathologic structure and function. Ophthalmology. 1982;89(6):531–590. [CrossRef] [PubMed]
Doi M Ning M Semba R Uji Y Refojo MF . Histopathologic abnormalities in rabbit retina after intravitreous injection of expansive gases and air. Retina. 2000;20(5):506–513. [CrossRef] [PubMed]
Kim EK Cristol SM Geroski DH McCarey BE Edelhauser HF . Corneal endothelial damage by air bubbles during phacoemulsification. Arch Ophthalmol. 1997;115(1):81–88. [CrossRef] [PubMed]
Hong A Caldwell MC Kuo AN Afshari NA . Air bubble-associated endothelial trauma in descemet stripping automated endothelial keratoplasty. Am J Ophthalmol. 2009;148(2):256–259. [CrossRef] [PubMed]
Sztarbala T Gos R Kedziora J Blaszczyk J Sibinska E Goralczyk M . [Changes in the antioxidant system of the aqueous humor, lens and erythrocytes after sulfur hexafluoride application to the vitreous of rabbits]. Klin Oczna. 1998;100(2):73–75. [PubMed]
Norn MS . Corneal thickness after cataract extraction with air in the anterior chamber. Acta Ophthalmol (Copenh). 1975;53(5):747–750. [CrossRef] [PubMed]
Irvine AR Kratz RP O'Donnell JJ . Endothelial damage with phacoemulsification and intraocular lens implantation. Arch Ophthalmol. 1978;96(6):1023–1026. [CrossRef] [PubMed]
Hirst LW Snip RC Stark WJ Maumenee AE . Quantitative corneal endothelial evaluation in intraocular lens implantation and cataract surgery. Am J Ophthalmol. 1977;84(6):775–780. [CrossRef] [PubMed]
Binkhorst CD Nygaard P Loones LH . Specular microscopy of the corneal endothelium and lens implant surgery. Am J Ophthalmol. 1978;85(5 pt 1):597–605. [CrossRef] [PubMed]
Bourne WM Brubaker RF O'Fallon WM . Use of air to decrease endothelial cell loss during intraocular lens implantation. Arch Ophthalmol. 1979;97(8):1473–1475. [CrossRef] [PubMed]
Sugar A Fetherolf EC Lin LL Obstbaum SA Galin MA . Endothelial cell loss from intraocular lens insertion. Ophthalmology. 1978;85(4):394–399. [CrossRef] [PubMed]
Forstot SL Blackwell WL Jaffe NS Kaufman HE . The effect of intraocular lens implantation on the corneal endothelium. Trans Sect Ophthalmol Am Acad Ophthalmol Otolaryngol. 1977;83(2):195–203. [PubMed]
Cheng H Sturrock GD Rubinstein B Bulpitt CJ . Endothelial cell loss and corneal thickness after intracapsular extraction and iris clip lens implantation: a randomised controlled trial (interim report). Br J Ophthalmol. 1977;61(12):785–790. [CrossRef] [PubMed]
Figure 1.
 
Slit lamp and vital staining assessment. (AF) In vivo slit lamp images of air (first column; right eye) and SF6 (second column; left eye) bubbles 1, 5, and 10 days (top, center, and bottom rows, respectively) after injection. The air bubble occupied 30% (A), 5% (B), and 0% (C) and the SF6 bubble occupied 50% (D), 35% (E), and 0% (F) of the anterior chamber 1, 5, and 10 days after injection, respectively. The fibrin membrane floating at day 5 in the anterior chamber of the eye injected with SF6 (white arrow) had retracted toward the angle superiorly at day 10. (GL) Ex vivo stereo microscopy images of the alizarin red and trypan blue-stained corneal endothelium of the same animal 10 days after the injection of air in the right eye (third column) and SF6 in the left eye (fourth column). The superior (first row), central (second row), and inferior (third row) corneal endothelial areas are shown (G, 2632 cells/mm2; H, 2688 cells/mm2; I, 2558 cells/mm2; J, 1527 cells/mm2; K, 2342 cells/mm2; L, 2717 cells/mm2). Scale bars: 100 μm (GL).
Figure 1.
 
Slit lamp and vital staining assessment. (AF) In vivo slit lamp images of air (first column; right eye) and SF6 (second column; left eye) bubbles 1, 5, and 10 days (top, center, and bottom rows, respectively) after injection. The air bubble occupied 30% (A), 5% (B), and 0% (C) and the SF6 bubble occupied 50% (D), 35% (E), and 0% (F) of the anterior chamber 1, 5, and 10 days after injection, respectively. The fibrin membrane floating at day 5 in the anterior chamber of the eye injected with SF6 (white arrow) had retracted toward the angle superiorly at day 10. (GL) Ex vivo stereo microscopy images of the alizarin red and trypan blue-stained corneal endothelium of the same animal 10 days after the injection of air in the right eye (third column) and SF6 in the left eye (fourth column). The superior (first row), central (second row), and inferior (third row) corneal endothelial areas are shown (G, 2632 cells/mm2; H, 2688 cells/mm2; I, 2558 cells/mm2; J, 1527 cells/mm2; K, 2342 cells/mm2; L, 2717 cells/mm2). Scale bars: 100 μm (GL).
Figure 2.
 
Slit lamp parameters, IOP, and central pachymetry values from day 0 to day 10. (A) Percentage of the anterior chamber occupied by the bubble. (B) Anterior chamber cells. (C) Anterior chamber flare. (D) Intraocular pressure. (E) Central corneal thickness. Slit lamp measurements were obtained daily on all eyes (n = 16 animals with complete data sets for air and SF6 from day 0 to day 10), whereas central pachymetry complete data sets could be obtained in only four animals (ultrasound pachymetry is not possible in the presence of a retrocorneal bubble). Error bars indicate SEM.
Figure 2.
 
Slit lamp parameters, IOP, and central pachymetry values from day 0 to day 10. (A) Percentage of the anterior chamber occupied by the bubble. (B) Anterior chamber cells. (C) Anterior chamber flare. (D) Intraocular pressure. (E) Central corneal thickness. Slit lamp measurements were obtained daily on all eyes (n = 16 animals with complete data sets for air and SF6 from day 0 to day 10), whereas central pachymetry complete data sets could be obtained in only four animals (ultrasound pachymetry is not possible in the presence of a retrocorneal bubble). Error bars indicate SEM.
Figure 3.
 
Preinjection and postinjection endothelial cell morphometric analysis of specular microscopy photos (n = 14 complete data sets). The mean of day 7 and day 10 values are reported for postinjection values. (A) Cell density. (B) Average cell area. (C) Coefficient of variation of cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 3.
 
Preinjection and postinjection endothelial cell morphometric analysis of specular microscopy photos (n = 14 complete data sets). The mean of day 7 and day 10 values are reported for postinjection values. (A) Cell density. (B) Average cell area. (C) Coefficient of variation of cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 4.
 
Postmortem endothelial cell morphometric analysis after vital staining. Measurements were taken in three corneal positions (superior, central, and inferior) (n = 15 complete data sets). (A) Cell density. (B) Average endothelial cell area. (C) Coefficient of variation in cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 4.
 
Postmortem endothelial cell morphometric analysis after vital staining. Measurements were taken in three corneal positions (superior, central, and inferior) (n = 15 complete data sets). (A) Cell density. (B) Average endothelial cell area. (C) Coefficient of variation in cell area. (D) Percentage of hexagonal cells. Error bars indicate SEM.
Figure 5.
 
Scanning electron microscopy. Two patterns of endothelial damage observed in the superior cornea of animals exposed to SF6 in one eye (left) and air in the other eye (right). Each row illustrates a pair of mate corneas. Damage was either similarly minimal (A, B) or significantly more important (C, D) in the eye injected with SF6 than in the mate eye injected with air. Endothelial cell damage included cell membrane disruption and missing cells. Patches of inflammatory cells scattered on the posterior corneal surface were occasionally seen (E). Scale bars: 50 μm (AD); 10 μm (E).
Figure 5.
 
Scanning electron microscopy. Two patterns of endothelial damage observed in the superior cornea of animals exposed to SF6 in one eye (left) and air in the other eye (right). Each row illustrates a pair of mate corneas. Damage was either similarly minimal (A, B) or significantly more important (C, D) in the eye injected with SF6 than in the mate eye injected with air. Endothelial cell damage included cell membrane disruption and missing cells. Patches of inflammatory cells scattered on the posterior corneal surface were occasionally seen (E). Scale bars: 50 μm (AD); 10 μm (E).
Figure 6.
 
Transmission electron microscopy. Pair of corneas of the same animal exposed to SF6 in one eye (A) and air in the other eye (B). In the cornea exposed to SF6, a nonspecific material lay in the subendothelial cell space. An inflammatory membrane, in which multiple cells such as monocytes, immature fibroblasts, and epithelioid histiocytes (keratic precipitates) were identified, covered the endothelium. No inflammatory membrane was found over the endothelial cells of cornea exposed to air. Scale bar: 2 μm (A, B).
Figure 6.
 
Transmission electron microscopy. Pair of corneas of the same animal exposed to SF6 in one eye (A) and air in the other eye (B). In the cornea exposed to SF6, a nonspecific material lay in the subendothelial cell space. An inflammatory membrane, in which multiple cells such as monocytes, immature fibroblasts, and epithelioid histiocytes (keratic precipitates) were identified, covered the endothelium. No inflammatory membrane was found over the endothelial cells of cornea exposed to air. Scale bar: 2 μm (A, B).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×