Experimental set-up
All experiments were performed on isolated porcine eyes in compliance with the ARVOstatement for the use of animals in ophthalmic research.
Porcine eyes were obtained within twenty four hours of the animal‟s death, transferred under aseptic conditions, and used within the same day after warming to 25oC using a saline bath. Use of tissue within 24 hours prevented corneal oedema, permitting good visualization of the anterior chamber. Lubricant gel (Methocel 2% Omnivision – Neuhausen) was applied to the surface of the cornea to prevent dehydration during the course of the experiment as required.
Under microscopic control, the anterior chamber of each eyewas cannulated at the limbus using a 30 gauge needle for infusion. After ensuring that the puncture site was watertight, the tip of the needle was positioned into the posterior chamber just behind the pupillary plane. The needle wasconnected via polyethylene tubing
to a microsyringe pump (SP200i, World Precision Instruments, Inc, Sarasota, FL, USA). The anterior chamber was then cannulated with a second needle, 25 gauge and water tight, which was connected viapolyethylene tubing to an electronic pressure transducer (Type BLPR, World Precision Instruments, Inc, Sarasota, FL, USA). Pressure measurements were then amplifiedand displayed on a pressure monitor (Ape BP-1,World Precision Instruments, Sarasota, FL, USA) and
transmitted to a data acquisition system (LabTrax - World Precision Instruments, Inc, Sarasota, FL, USA). Data were saved on a computer and printed. Thesyringe pump-pressuretransducer system was a closed watertight system. All tubes were filled with Dulbecco's phosphate-buffered salt solution (PBS) (Grand Island
Biological Co., Grand Island, N.Y.) and air bubbles removed. The intraocular pressure was measured in mmHg on thechart recorder, which was calibrated against a column of water before the experiment (Figures 21, 22).
Figure 21. Photograph of the experimental setup.
Figure 22. Scheme of the experimental setup.
Needle diameters and canal lengths
10 eyes were tested for each combination of needle diameter and canal length (6 combinations), for a total of 60 experimental eyes. An additional 10 eyes were
controls in which no scleral canal was created.
The needles diameters and scleral canal lengths studied are outlined in the table 7.
Group number
Eyes per group
Needle size (g)
Canal
length (mm)
0 10 No injection
1 10 27 4
2 10 27 6
3 10 23 4
4 10 23 6
5 10 21 4
6 10 21 6
Table 7. Needles diameters and scleral canal lengths studied in this experiment.
Outflow facility measurements
After cannulation of the anterior chamber, the inflow rate was adjusted to give an intraocular pressure of 10 mmHg. The inflow rate was then increased until the intraocular pressure stabilized at 20 mmHg. The infusion rate was then altered again until a stable pressure of 30 mmHg was attained. All values were
continuously recorded during the experiments.
The infusion measurements were plotted against the intraocularpressures.The outflow facility (C) was calculated with the Goldmannequation:
C=I/IOP
I= I2-I1 where I1 and I2 are successive inflow rates (µl/min)
IOP= P2–P1 where P1 and P2 represent IOP at I1 and I2 respectively (mmHg)
NASHA gel ab exerno intrascleral injection
Once pressure was stabilised at 10 mmHg, a needle connected to the NASHA™
gel syringe entered the subconjunctival space 4 or 6 mm from the limbus to penetrate the sclera and is 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 (4mm or 6 mm) was measured with a surgical calliper.
Then the needle was slowly withdrawn as NASHA™ gel was injected
intrasclerally to create a scleral canal in the needle tract, 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 using the Shapiro-Wilk test. T-test was used to compare the group of eyes injected with NASHA™ gel (60 eyes) with the control group (10 eyes) for both 10 to 20 mmHg and 20 to 30 mmHg pressure change and to construct a 95% confidence interval 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 (6
subgroups of 10 eyes for both 10 to 20 mmHg and 20 to 30 mmHg pressure change).
Statistical significance was defined at 5%. Results are presented as mean ± standard deviation.
RESULTS
Mean outflow facility for each group and at each pressure level are presented in table 8. A significant increase in outflow facility was found between the control
group and the NASHA™ injected group of 60 eyes for both 10-20 mmHg and 20-30 mmHg pressure change. Increase in outflow facility was independent of the canal lengths and the needle sizes used for both 10-20 mmHg (p=0.82) and 20-30 mmHg (p=0.99) pressure change.
In two additional eyes, hypotony occurred after creation of a 4 mm canal, causing movements of and leakage around the infusion needle so these eyes were not used in the experiment.
Figure 23. Outflow facility for 10-20 mmHg pressure change.
Figure 24. Outflow facility for 20-30 mmHg pressure change.
Pressure change Table 8. Outflow facility of the control group and the injected group.
DISCUSSION
This is the first study 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 if the technique of NASHA gel canal creation is feasible in vitro and to try to optimize the best combination of length canal and diameter in this model. The outflow facility of a recently enucleated eye is very close to that in vivo, so this should be a good experimental model114.
This experiment was performed in an inert tissue, which cannot stimulate any inflammatory reaction or wound healing process. Consequently, potential cicatrisation of the intrascleral canal cannot be determined with our experimental model. The long term efficacy of this procedure remains to be determined in vivo.
Hyaluronic acid is a uniform, unbranched, linear polysaccharide with the same simple chemical structure in all species and tissues. NASHA gel is synthesized from a non-animal 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 which can take any form. This NASHA gel is highly permeable to aqueous humor99.
In this experiment, a single intrascleral channel into the anterior chamber was created with a 21G or 23G or 27G needle for 4mm or 6mm 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 10-20 mmHg and 20-30 mmHg pressure change. This is not unexpected considering that a single hole of 12 μm has been calculated to be sufficient to provide a normal outflow facility115.
Although one might have expected that with larger needle diameters and the 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 drops 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 drops in outflow facility.
While outflow facility was improved in this very short term in vitro study, many additional issues remain to be overcome before in vivo success of this procedure is demonstrated, including long term patency of the gel filled scleral canal in vivo,
absence of inflammatory reaction, and long term increase in facility of outflow in vivo.
This technique is minimally invasive, leaving a large area of intact conjunctiva and sclera should trebeculectomy or other surgeries were to 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 almost up to a year99. The NASHA gel has been found to stay in the intrascleral canal of a rabbit eye for at least 16 weeks (figures 25,26).
Our hypothesis is that NASHA gel stays in scleral canals for a significant period of time 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 25 and Figure 26 show an intrascleral gel canal 16 weeks after an 18G needle NASHA gel intrascleral injection between 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 Q-Med). Figure 25 under low magnification shows a cross-section of the intrascleral canal. In the Figure 26 under higher magnification, light grey material is visible in the canal which is some of the injected gel. 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 inflammatory reaction is notably absent, a sign that the scleral tissue tolerated the injected NASHA gel very well.
While these data support our hypothesis, these results need to be confirmed in further in vivo studies.
Figure 25. Cross-section of an intrascleral canal 16 weeks after an 18G needle NASHA™
gel intrascleral injection between superior and lateral rectus in an in vivo rabbit eye (Image courtesy of Ulf Stenevi, Uppsala, Sweden).
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 cadaver porcine eyes, we had very little difficulty performing the intrascleral injections Reproducibility problems were rare. As we have already mentioned, only two cases
of hypotony occurred after hyaluronic gel injection with a 4mm canal. NASHA gel can be coloured to facilitate its visualization and help avoid intracameral injection.
Figure 26. Higher magnification of the intrascleral canal presented in figure 10. Note the light grey material in the canal which is some of the injected gel (Image courtesy of Ulf Stenevi, Uppsala, Sweden).
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. Only two cases of hypotony occurred after hyaluronic gel injection with a 4mm canal. We believe that greater experience with this technique will minimize this problem.
CONCLUSION
A single ab externo intrascleral injection of stabilized, non-animal, hyaluronic acid increases the outflow facility in isolated porcine eyes. This appears to be potentially a promising new technique for lowering intraocular pressure. It remains to be seen if
positive results can be reproduced with this simple procedure for significant periods of time in humans.
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