August 2009
Volume 50, Issue 8
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Glaucoma  |   August 2009
Outflow Facility in Isolated Porcine Eyes after Creation of an Intrascleral Canal by Injection of Stabilized Hyaluronic Acid
Author Affiliations
  • Nikolaos Mavrakanas
    From the Glaucoma Surgery Research Laboratory, Ophthalmology Department, Geneva University Hospitals, Geneva, Switzerland.
  • Tarek Shaarawy
    From the Glaucoma Surgery Research Laboratory, Ophthalmology Department, Geneva University Hospitals, Geneva, Switzerland.
Investigative Ophthalmology & Visual Science August 2009, Vol.50, 3759-3762. doi:10.1167/iovs.08-2801
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      Nikolaos Mavrakanas, Tarek Shaarawy; Outflow Facility in Isolated Porcine Eyes after Creation of an Intrascleral Canal by Injection of Stabilized Hyaluronic Acid. Invest. Ophthalmol. Vis. Sci. 2009;50(8):3759-3762. doi: 10.1167/iovs.08-2801.

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

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Abstract

purpose. To investigate outflow facility in isolated porcine eyes after creation of an intrascleral canal by injection of stabilized, nonanimal, hyaluronic acid gel (NASHA; Q-Med AB, Uppsala, Sweden).

methods. Outflow facility of 60 porcine eyes was measured after creation of an intrascleral canal into the anterior chamber by injection of NASHA gel using six different combinations of needle size (21, 23, and 27 gauge) and canal length (4.6 mm). Ten eyes were tested in each of the six subgroups and an additional 10 were control eyes. After cannulation of the anterior chamber and adjustment of the inflow rate to achieve a stable pressure of 10 mm Hg, an intrascleral channel was created by injection of NASHA gel. The inflow rate was then increased to achieve a stable pressure of 20 mm Hg and then again adjusted to maintain a stable pressure of 30 mm Hg.

results. A significant increase in outflow facility was found between the control group and the NASHA gel–injected group (60 eyes) for both the 10- to 20-mm Hg and the 20- to 30-mm Hg pressure change (P < 0.001). Increase in outflow facility was independent of the canal lengths and the needle sizes used for both the 10- to 20-mm Hg (P = 0.82) and 20- to 30-mm Hg (P = 0.99) pressure change.

conclusions. A single ab externo intrascleral canal created by injection of NASHA gel increases the outflow facility in isolated porcine eyes. This is a potentially promising new technique for lowering intraocular pressure. It remains to be seen whether these positive results can be reproduced for significant periods in humans.

Surgical techniques for lowering intraocular pressure (IOP) have evolved since the first trabeculectomy was performed in the late 1960s. 1 2 Nonpenetrating glaucoma surgery 3 and shunt tubes have been shown to be useful in the surgical treatment of glaucoma 4 ; however, trabeculectomy remains the procedure of choice for most surgeons around the world. 5 The long-term efficacy of trabeculectomy and tube shunts, as well as the complications 6 7 and a suboptimal degree and longevity of IOP-reduction with less invasive techniques, 8 indicate the need for new, safer, effective glaucoma surgery. Moreover, a safe and simple surgical treatment would be an attractive option for patients burdened by expensive medical glaucoma treatment, those with compliance problems, and those in developing countries for whom medical treatments are unavailable or inaccessible. 9  
In this study, we measured outflow facility in isolated porcine eyes after creation of an intrascleral canal by injection of stabilized, nonanimal hyaluronic acid (NASHA; Q-Med AB, Uppsala, Sweden) using several combinations of intrascleral canal lengths and needle diameters. Aqueous humor passes though the NASHA gel in the scleral canal, creating a new aqueous outflow pathway. 
The goal of the study was to see whether the NASHA gel scleral canal technique is feasible in our in vitro model, to measure any change in outflow facility with this procedure, and to try to determine the best combination of canal length and diameter in relation to increased outflow facility, if any. 
Methods
Experimental Setup
Porcine eyes were obtained within 24 hours of the animal’s death, transferred under aseptic conditions, and used within the same day after warming to 25°C in a saline bath. Use of tissue within 24 hours prevented corneal edema, permitting good visualization of the anterior chamber. Lubricant gel (methocel 2%; Omnivision, Neuhausen, The Netherlands) was applied to the surface of the cornea, as required, to prevent dehydration during the course of the experiment. 
For the infusion, the anterior chamber of each eye was cannulated under microscopic control at the limbus with a 30-gauge needle. The puncture site was determined to be watertight, and the tip of the needle was positioned in the posterior chamber just behind the pupillary plane. The needle was connected via polyethylene tubing to a microsyringe pump (SP 200i; World Precision Instruments, Inc., Sarasota, FL). The anterior chamber was then cannulated with a second needle, 25-gauge and water tight, which was connected via polyethylene tubing to an electronic pressure transducer (Type BLPR; World Precision Instruments, Inc.). Pressure measurements were then amplified and displayed on a pressure monitor (Ape BP-1; World Precision Instruments) and transmitted to a data-acquisition system (LabTrax; World Precision Instruments, Inc.). Data were saved on a computer and printed. The syringe pump–pressure transducer system was a closed, watertight system. All tubes were filled with Dulbecco’s phosphate-buffered salt solution (PBS; Invitrogen-GIBCO, Grand Island, NY) and the air bubbles removed. The intraocular pressure was measured in millimeters of mercury on the chart recorder, which was calibrated against a column of water before the experiment (Fig. 1)
The needle diameters and scleral canal lengths are outlined in Table 1
Ten eyes were tested for each combination of needle diameter and canal length (six combinations), for a total of 60 experimental eyes. An additional 10 eyes were control samples in which no scleral canal was created. 
Outflow Facility Measurements
After cannulation of the anterior chamber, the inflow rate was adjusted to give an intraocular pressure of 10 mm Hg. The inflow rate was then increased until the intraocular pressure stabilized at 20 mm Hg. The infusion rate was then altered again until a stable pressure of 30 mm Hg was attained. All values were continuously recorded during the experiments. 
The infusion measurements were plotted against the intraocular pressures. The outflow facility (C) was calculated with the Goldmann equation:  
\[C\ {=}\ {\Delta}I/{\Delta}\mathrm{IOP}\]
where  
\[{\Delta}I\ {=}\ I_{2}\ {-}\ I_{1}\]
where I 1 and I 2 are successive inflow rates (microliters per minute), and  
\[{\Delta}\mathrm{IOP}\ {=}\ P_{2}\ {-}\ P_{1}\]
where P 1 and P 2 represent IOP at I 1 and I 2, respectively (mm Hg). 
NASHA Gel Ab Externo Intrascleral Injection
Once pressure was stabilized at 10 mm Hg, a needle connected to the NASHA gel syringe entered the subconjunctival space 4 or 6 mm from the limbus to penetrate the sclera and was then passed intrasclerally to form a scleral canal into the anterior chamber with the exit point in the trabecular meshwork. The length of the scleral canal (4 or 6 mm) was measured with a surgical caliper. 
The needle was slowly withdrawn as the NASHA gel was injected intrasclerally to create a scleral canal in the needle tract, while avoiding injection into the anterior chamber. Six combinations of canal length and diameter were tested in the experiments, as outlined in Table 1 . Only one injection of NASHA gel was used per eye. 
Statistical Analysis
Normality was verified with the Shapiro-Wilk test. The t-test was used to compare the group of eyes injected with NASHA gel (60 eyes) with the control group (10 eyes) for both the 10- to 20-mm Hg and the 20- to 30-mm Hg pressure change and to construct a 95% confidence interval (CI) for the difference between the means of the two groups. One-way analysis of variance (ANOVA) was used to look for differences in outflow facility between the subgroups of different needle diameter and canal length (six subgroups of 10 eyes for both pressure changes). Statistical significance was defined at 5%. Results are presented as the mean ± SD. 
Results
The mean outflow facility for each group and at each pressure level is presented in Table 2 . A significant increase in outflow facility was found between the control group and the NASHA-injected group of 60 eyes for both the 10- to 20-mm Hg and the 20- to 30-mm Hg pressure changes. Increase in outflow facility was independent of the canal lengths and the needle sizes used for the 10- to 20-mm Hg (P = 0.82) and the 20- to 30-mm Hg (P = 0.99) pressure changes. The distribution of results for each of the categories tested is shown in Table 3
In two additional eyes, hypotony occurred after creation of a 4 mm canal, causing movements of and leakage around the infusion needle, so that these eyes were not used in the experiment. 
Discussion
This is the first report of a new, simple surgical technique for creating a microfistula to lower intraocular pressure. In this in vitro porcine eye study, we investigated outflow facility alterations after creation of a single intrascleral NASHA gel canal. The basic goal of this study was to see whether the technique of NASHA gel canal creation is feasible in vitro and to try to optimize the best combination of length of canal and diameter in this model. The long-term efficacy of this procedure cannot be determined with our experimental model and remains to be determined in vivo. 
The outflow facility of a recently enucleated eye is very close to that in vivo, and so this should be a good experimental model. 10  
Hyaluronic acid is a uniform, unbranched, linear polysaccharide (Fig. 2A)with the same simple chemical structure in all species and tissues. NASHA gel is synthesized from a nonanimal source avoiding potentially harmful antigenic proteins, viruses, and prions. Stabilization involves polymerization of the hyaluronic acid molecules in solution to form a viscous, very cohesive, hyaluronic acid gel that can take any form (Figs. 2B 2C)and is highly permeable to aqueous humor. 11  
NASHA has been successfully used for treatment of knee and hip osteoarthritis, 12 13 14 for facial tissue augmentation, 15 16 17 for treatment of vesicoureteral reflux in children, 18 19 20 and for treatment of urinary stress incontinence in women. 21 22 23  
In this experiment, a single intrascleral channel into the anterior chamber was created with a 21-, 23- or 27-gauge needle for a 4- or 6-mm intrascleral canal length. A significant increase in outflow facility was found between the control group (10 eyes) and the NASHA gel-injected group. However, the increased outflow facility did not depend on canal length or needle diameter for both the 10- to 20-mm Hg and the 20- to 30-mm Hg pressure changes. This finding is not unexpected, considering that a single hole of 12 μm has been calculated to be sufficient to provide normal outflow facility. 24  
Although one might have expected that with larger needle diameter and shorter canal length, we would have found lower pressures, only a trend in this direction was observed. Perhaps the presence of the gel in the canal acts to prevent large decreases in outflow facility. Second, the difference in diameters in these canals in this in vitro model may not have been large enough to cause a significant difference in outflow facility. Third, the diameter of the beginning of the canal at the scleral entrance and/or the diameter of the canal as it enters the trabecular meshwork may not be fully maintained by the gel at those locations, thus limiting large decreases in outflow facility. 
Although outflow facility was improved in this very short-term in vitro study, many additional problems remain to be overcome before the success of this procedure is demonstrated in vivo, including long-term patency of the gel-filled scleral canal, absence of inflammatory reaction, and long-term increase in outflow facility. 
This technique is minimally invasive, leaving a large area of intact conjunctiva and sclera should trabeculectomy or other surgery be necessary subsequently. A second intrascleral injection of the NASHA gel could be performed concomitantly or later if target pressure were not attained. 
The half-life of endogenous hyaluronic acid is short in most tissues, varying from half a day to a few days. NASHA gel has an increased half-life and has been found to remain in the skin for many months, sometimes even up to a year. 11 The NASHA gel has been found to stay in the intrascleral canal of a rabbit eye for at least 16 weeks (Fig. 3)
Our hypothesis is that NASHA gel stays in scleral canals for a significant period maintaining patency long enough to allow endothelial cells to line the scleral channel, creating a microfistula from the anterior chamber to the subconjunctival space to form a shallow bleb. NASHA gel could also be injected into the subconjunctival space to help form a bleb and help avoid subconjunctival scarring. 
Figure 3shows an intrascleral gel canal 16 weeks after an 18-gauge needle NASHA gel intrascleral injection between the superior and lateral rectus in an in vivo rabbit eye. The injection was performed with the same technique used in our porcine eye experiment. These data have not been previously published (courtesy of Ulf Stenevi, Uppsala, Sweden). Figure 3A , under low magnification, shows a cross section of the intrascleral canal. In Figure 3Bunder higher magnification, light gray injected gel is visible in the canal. It is very difficult to keep all the implanted gel in place on the slide during fixation and staining, but some of it is still visible. Note that the canal is lined with endothelial-like cells and an inflammatory reaction is notably absent, a sign that the scleral tissue tolerated the injected NASHA gel very well. Although these data support our hypothesis, the results must be confirmed in vivo. 
This surgical procedure is simple and the learning curve short. A needle connected to the NASHA gel syringe enters the subconjunctival space 4 or 6 mm from the limbus to penetrate the sclera and is passed intrasclerally to form a scleral canal into the anterior chamber. The NASHA gel is then injected into the scleral canal as the needle is withdrawn. After practicing the injection technique in 10 porcine eyes, we had very little difficulty in performing the intrascleral injections. Reproducibility problems were rare. As already mentioned, only two cases of hypotony occurred after hyaluronic gel injection with a 4-mm canal. NASHA gel can be colored to facilitate its visualization and help avoid intracameral injection. 
The NASHA gel in the intrascleral canal acts as a mechanical barrier to protect against a significant drop in outflow resistance which could result in hypotony and hypotony-related complications. Considering that only two cases of hypotony occurred after hyaluronic gel injection with a 4-mm canal, we believe that greater experience with this technique will minimize this problem. 
Conclusion
A single ab externo intrascleral injection of stabilized, nonanimal, hyaluronic acid increases the outflow facility in isolated porcine eyes. This technique appears to have potential for lowering intraocular pressure. It remains to be seen whether positive results can be reproduced with this simple procedure for significant periods in humans. 
 
Figure 1.
 
(A) Photograph of the experimental setup. (B) Schematic of the experimental setup.
Figure 1.
 
(A) Photograph of the experimental setup. (B) Schematic of the experimental setup.
Table 1.
 
Needle Diameters and Scleral Canal Lengths
Table 1.
 
Needle Diameters and Scleral Canal Lengths
Subgroups Needle Diameter (Gauge) Canal Length (mm)
1 27 4
2 27 6
3 23 4
4 23 6
5 21 4
6 21 6
Table 2.
 
Outflow Facility of the Study Groups
Table 2.
 
Outflow Facility of the Study Groups
Pressure Change (mm Hg) Control Group Injected Group P 95% CI (Difference of the Means)
10–20 0.99 ± 0.13 1.20 ± 0.21 <0.001 0.11–0.32
20–30 1.07 ± 0.19 1.28 ± 0.18 0.001 0.08–0.33
Figure 4.
 
Results for Each of the Categories Tested
Figure 4.
 
Results for Each of the Categories Tested
Figure 2.
 
(A) Chemical structure of NASHA. (B, C) Different forms of the NASHA gel.
Figure 2.
 
(A) Chemical structure of NASHA. (B, C) Different forms of the NASHA gel.
Figure 3.
 
(A) Cross section of an intrascleral canal 16 weeks after an 18-gauge needle NASHA gel intrascleral injection between superior and lateral rectus in an in vivo rabbit eye. (B) Higher magnification of the intrascleral canal. Note the light gray material in the canal, which is some of the injected gel. (Image courtesy of Ulf Stenevi, Uppsala, Sweden.)
Figure 3.
 
(A) Cross section of an intrascleral canal 16 weeks after an 18-gauge needle NASHA gel intrascleral injection between superior and lateral rectus in an in vivo rabbit eye. (B) Higher magnification of the intrascleral canal. Note the light gray material in the canal, which is some of the injected gel. (Image courtesy of Ulf Stenevi, Uppsala, Sweden.)
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Figure 1.
 
(A) Photograph of the experimental setup. (B) Schematic of the experimental setup.
Figure 1.
 
(A) Photograph of the experimental setup. (B) Schematic of the experimental setup.
Figure 4.
 
Results for Each of the Categories Tested
Figure 4.
 
Results for Each of the Categories Tested
Figure 2.
 
(A) Chemical structure of NASHA. (B, C) Different forms of the NASHA gel.
Figure 2.
 
(A) Chemical structure of NASHA. (B, C) Different forms of the NASHA gel.
Figure 3.
 
(A) Cross section of an intrascleral canal 16 weeks after an 18-gauge needle NASHA gel intrascleral injection between superior and lateral rectus in an in vivo rabbit eye. (B) Higher magnification of the intrascleral canal. Note the light gray material in the canal, which is some of the injected gel. (Image courtesy of Ulf Stenevi, Uppsala, Sweden.)
Figure 3.
 
(A) Cross section of an intrascleral canal 16 weeks after an 18-gauge needle NASHA gel intrascleral injection between superior and lateral rectus in an in vivo rabbit eye. (B) Higher magnification of the intrascleral canal. Note the light gray material in the canal, which is some of the injected gel. (Image courtesy of Ulf Stenevi, Uppsala, Sweden.)
Table 1.
 
Needle Diameters and Scleral Canal Lengths
Table 1.
 
Needle Diameters and Scleral Canal Lengths
Subgroups Needle Diameter (Gauge) Canal Length (mm)
1 27 4
2 27 6
3 23 4
4 23 6
5 21 4
6 21 6
Table 2.
 
Outflow Facility of the Study Groups
Table 2.
 
Outflow Facility of the Study Groups
Pressure Change (mm Hg) Control Group Injected Group P 95% CI (Difference of the Means)
10–20 0.99 ± 0.13 1.20 ± 0.21 <0.001 0.11–0.32
20–30 1.07 ± 0.19 1.28 ± 0.18 0.001 0.08–0.33
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