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Keywords:

  • epinephrine;
  • intracameral mydriatics;
  • isoprenaline;
  • mydriatics;
  • phenylephrine

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose:  To compare the mydriatic effect of intracamerally injected isoprenaline plus phenylephrine to phenylephrine alone and to epinephrine in a porcine eye model, aiming to eventually find the best combination of adrenergic substances for surgical mydriasis in humans.

Methods:  In this study, we used 89 intact eyes from newly slaughtered pigs, pretreated with 2.0 mg of intracameral acetylcholine. After waiting 60 seconds for miosis to develop, 0.15 ml 0.3% isoprenaline and 0.15 ml 3.0% phenylephrine were injected sequentially with a 90-second interval in 21 eyes. In another 22 eyes, the same substances were given in the reverse order. In 20 eyes, 0.15 ml of 0.025% epinephrine was injected, and as a negative control 0.15 ml of balanced salt solution was injected in 26 eyes. The pupils were filmed during the treatments, and the mean pupil diameters were measured every 15 seconds from the video recordings.

Results:  Phenylephrine injected after isoprenaline had a larger mydriatic effect than epinephrine (p < 0.01). Without isoprenaline pretreatment, the mydriatic effect of phenylephrine was significantly smaller than that of epinephrine (p < 0.05). Isoprenaline also exhibited a small mydriatic effect of its own.

Conclusions:  The β-receptor stimulator isoprenaline enhances the mydriatic effect of intracameral phenylephrine, indicating a role for the β-receptor in the mydriatic response. Mydriasis mediated by β-receptors may explain why nonspecific adrenergic stimulators such as epinine and epinephrine can have larger mydriatic effects than the specific α1-receptor stimulator phenylephrine.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The increased use of intracameral mydriatics in intraocular surgery has given us a possibility to use other mydriatic substances than the traditional anticholinergics and α1-adrenergics (Cionni et al. 2003; Lundberg & Behndig 2009; Myers & Shugar 2009), because the doses can be reduced, and we no longer have to consider limitations in bioavailability because of the corneal barrier. Recently, intracameral mydriatics have come into focus because of their benefits in the so-called intraoperative floppy iris syndrome (Gurbaxani & Packard 2007; Masket & Belani 2007). To obtain mydriasis for example in cataract surgery, we ourselves routinely use an intracameral injection of 150 μl of a solution containing lidocaine 1% and phenylephrine 1.5% at the start of the surgical procedure. This regimen has proved reliable and safe (Lundberg & Behndig 2003; Behndig & Eriksson 2004), but gives a slightly weaker mydriatic effect than topical regimens involving 10% phenylephrine and 1% cyclopentolate (Lundberg & Behndig 2003). Pharmacologically, phenylephrine ((R)-3-[-1-hydroxy-2-(methylamino)ethyl]phenol) is a specific α1-receptor agonist, and it is generally accepted that α1-receptors are the main mediators of the adrenergic mydriatic response of the iris dilator (Soldati et al. 1993; Yu & Koss 2002, 2003). Mydriasis from an adrenergic substance, however, may also in part owe to stimulation of iris β-receptors, resulting in a relaxation of the pupil sphincter (Patil 1969; Yoshitomi et al. 1988; Patil & Weber 1991; Geyer et al. 1998; Toda et al. 1999; Barilan et al. 2003). This additional effect may explain why epinephrine ((R)-4-(1-hydroxy-2-(methylamino)ethyl)benzene-1,2-diol), a nonselective agonist of the adrenergic receptors (α1, α2, β1, β2 and β3), is a more potent mydriatic agent than phenylephrine when injected intracamerally in humans (Myers & Shugar 2009). In accordance with this finding, we earlier demonstrated that the nonselective adrenergic stimulator epinine is superior to phenylephrine in creating mydriasis in a porcine eye model (Lundberg & Behndig 2009).

To assess the exact contributions from different receptors on the adrenergic mydriatic response, however, more specific stimulators should be employed. In this study, we used isoprenaline (4-[1-hydroxy-2-(isopropylamino)ethyl]benzene-1,2-diol) to specifically assess the effect of the β-receptors. Isoprenaline is a β-receptor agonist, structurally similar to epinephrine but acting selectively on the β-receptors, stimulating β1 and β2 receptors equally.

The aim of this study was to further explore the mechanisms underlying adrenergic mydriasis by comparing a combination of the β-stimulator isoprenaline and the α1-stimulator phenylephrine to phenylephrine alone or the more unspecific adrenergic stimulator epinephrine in a post-mortem porcine eye model.

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The research ethics committee of Umeå University, Umeå, Sweden, approved this study, and the study was carried out at the surgical laboratory of Advanced Medical Optics, Inc., in Uppsala, Sweden. For this study, a total of 89 newly slaughtered porcine eyes were used. Within 24–48 hr after death, the eyes were delivered from a local slaughterhouse and kept on ice until just before the investigation, when they were warmed to ambient temperature. All 89 eyes were pretreated with 2.0 mg of intracameral acetylcholine (0.2 ml 1% Miochol®-E; Novartis Ophtalmics, Inc. Täby, Sweden) to contract the pupil. After waiting 60 seconds for miosis to develop, in the first treatment group, 0.15 ml 0.3% isoprenaline and 0.15 ml 3.0% phenylephrine were injected sequentially in the mentioned order with a 90-second interval (n = 21). In the second group (n = 22), 0.15 ml 3.0% phenylephrine was injected firstly, followed by 0.15 ml 0.3% isoprenaline after 90 seconds. In the third group, 0.15 ml of 0.025% epinephrine was injected (n = 20). The doses and concentrations of the substances were adapted from human clinical studies (Myers & Shugar 2009) (Lundberg & Behndig 2003; Behndig & Lundberg 2010) or in the case of isoprenaline, determined in a small pilot series. As a negative control, 0.15 ml of balanced salt solution (BSS; Alcon Laboratories, Inc., Fort Worth, TX, USA) was injected (n = 26). In all four groups, the pupils were filmed with a digital video camera connected to an operation microscope during an observation period of 90 seconds, and a masked observer measured the smallest and the largest perpendicular pupil diameters from the video recordings, using the filmed blade of a 2.5-mm-slit knife as a calibration reference. The mean pupil diameter was calculated for each eye before each injection and 15, 30, 45, 60 and 90 seconds after the injection, and the mean pupil diameter from the negative control group at the corresponding time point was subtracted from all values.

The isoprenaline, phenylephrine and epinephrine hydrochloride solutions were prepared from stem solutions of the respective substances, using BSS (Alcon Laboratories, Inc.) as a dilutant. The stem solutions were prepared by the Product and Laboratory Department of the Swedish Pharmacy (Apoteksbolaget AB) from sterile salts of the respective substances.

Student′s two-tailed or paired t-test was used for statistical comparison. The results are presented as means ± standard deviations. Bonferroni corrections were performed when comparing more than two groups, and a p-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phenylephrine injected after isoprenaline rendered a significantly larger mydriatic effect than phenylephrine injected firstly (p < 0.05 after Bonferroni correction from 30 to 45 seconds; Fig. 1). This effect was even larger than that of epinephrine (p < 0.05 after Bonferroni correction from 30 seconds and on; Fig. 1). The mydriatic effect of phenylephrine wore off slightly towards the end of the observation period, but not when phenylephrine was injected after isoprenaline (Fig. 1). Generally, however, phenylephrine and epinephrine showed a similar mydriatic time curve after intracameral injection in this model (Fig. 1). BSS showed no significant mydriatic effect (Fig. 1). Isoprenaline, however, did show a small, but significant mydriatic effect, whether injected before or after phenylephrine (p < 0.05 from 15 to 45 seconds when injected firstly; p < 0.05 at 15 seconds when injected secondly; Fig. 2).

image

Figure 1.  The increase in pupil size in porcine eyes after an intracameral injection of 0.15 ml 0.025% epinephrine (E; n = 20), balanced salt solution (BSS; n = 26), 0.3% isoprenaline followed by 3.0% phenylephrine (IP; n = 21) or the latter two in the reversed order (PI; n = 22), means and standard errors of the mean. The mydriatic effect of phenylephrine is significantly enhanced after pretreatment with isoprenaline (p < 0.05 from 30 to 60 seconds), making it even larger than that of epinephrine (p < 0.05 at 30 seconds; p < 0.01 at 45–90 seconds).

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image

Figure 2.  The increase in pupil size in porcine eyes after an intracameral injection of 0.3% isoprenaline (I(IP); n = 21) or 0.3% isoprenaline after 3.0% phenylephrine (I(PI); n = 22), means and standard errors of the mean. Isoprenaline has a significant mydriatic effect in both groups (p < 0.05 from 15 to 45 seconds for P(IP); p < 0.05 at 15 seconds for P(PI)).

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study, we demonstrate that phenylephrine injected after isoprenaline has a significantly larger mydriatic effect than after a primary injection and that the sum effect of isoprenaline plus phenylephrine effect is larger than that of epinephrine. This study is the first to demonstrate a greater mydriatic effect from intracameral phenylephrine when injected after isoprenaline compared to when injected alone, a mydriatic enhancement confirmed by a small but significant mydriatic effect from isoprenaline itself, when injected either before or after phenylephrine. From these findings, it appears that the relaxing effect on the pupil sphincter from isoprenaline is sufficiently strong to partially counteract the contracting effect from intracameral acetylcholine.

Our findings thus indicate that β-receptor stimulation enhances the mydriatic effect of the α1-receptor stimulator phenylephrine, indicating a role for β-receptors in the mydriatic response. It has previously been shown by others that stimulation of different iris β-receptors can cause a relaxation of the iris sphincter in various species (Patil 1969; Patil & Weber 1991; Geyer et al. 1998; Toda et al. 1999; Barilan et al. 2003). Likewise, our group has previously demonstrated that epinine, a more unspecific adrenergic stimulator, has a mydriatic effect superseding that of phenylephrine (Lundberg & Behndig 2009). It may well be that mydriasis mediated by β-receptors explains why nonspecific adrenergic stimulators such as epinine (Lundberg & Behndig 2009) or epinephrine, as shown by Myers & Shugar (2009), can have larger mydriatic effects than a specific α1-receptor stimulator.

In this study, it is noteworthy that because of logistic reasons, we were unable to receive the porcine eyes within 24 hr, as in our previous study (Lundberg & Behndig 2009). Therefore, the mydriatic responses in this study were generally smaller, but still fully measurable and possible to quantify and compare between the groups. All eyes in this study were pretreated with intracameral acetylcholine to induce miosis. This is necessary because of the absent natural pupil sphincter tonus post-mortem. It is worth mentioning that although our aim was to minimize any post-mortem changes, c.f. by and keeping the eyes on ice, weakening of the pupil dilator may have contributed to generally small mydriatic responses in our study. Another weak point in the design of this study may be that the genetic background of the animals may have differed from our previous study, and also within the investigations, contrary to when inbred and genetically controlled strains of laboratory animals are used. On the other hand, when differences between treatment groups are clearly demonstrated in a heterogeneous but randomized material such as this, the differences are probably more likely to be clinically relevant, as the setting resembles the clinical situation more closely in this respect.

We feel it is of great importance to run an investigation such as the present one during a short time frame, which we did. This has probably contributed to the fact that no differences in mydriatic response could be demonstrated between the eyes treated early in the batch, and those given the same treatment towards the end of the study (data not shown). Another important feature of this study was the use of a rather large control group, to rule out any unspecific effects on pupil size from injecting a volume of fluid into the anterior chamber. It can here clearly be demonstrated that no such effects occur in this porcine eye model.

When using an animal model, it remains to be shown that the findings are transferable to humans. The anterior segment of the porcine eye has an acceptable degree of anatomical and ultrastructural resemblance with the human eye (Bachmann et al. 2006), and isolated porcine eyes (Wikberg-Matsson et al. 2000; Wikberg-Matsson & Simonsen 2001) or isolated porcine iris muscles (Toda et al. 1999) are established models for investigating ocular adrenoceptors (Toda et al. 1999; Wikberg-Matsson et al. 2000; Wikberg-Matsson & Simonsen 2001). In addition, for an intracameral mydriatic to be usable in humans safety issues, possible adverse reactions et cetera also have to be addressed. Phenylephrine and epinephrine have already been used intracamerally in large numbers of patients and have been thoroughly evaluated for safety (Lundberg & Behndig 2003; Myers & Shugar 2009). Isoprenaline, however, has never been used intracamerally in humans. Although cardiovascular or other systemic side-effects are unlikely at the low doses required for intracameral use (Lundberg & Behndig 2003), the substance still has to be evaluated for ocular safety, particularly regarding the corneal endothelium. Generally, adrenergic substances are prepared at a low pH to prevent auto-oxidation. For intracameral use, a substance should have a pH of approximately 6.5–8.5 to avoid endothelial toxicity, and the substance also needs to be bisulphite free and isotonic. These criteria are met by the phenylephrine and epinephrine solutions currently in clinical use, but would also need to be met by an isoprenaline solution. In addition to all this, appropriate dilution and dosing for humans have yet to be established. These tasks are, however, beyond the scope of this basic effect evaluation and should be addressed in human trials.

To conclude, we here show in a porcine eye model that the β-receptor stimulator isoprenaline enhances the mydriatic effect of intracameral phenylephrine, which indicates a role for the β-receptor in the mydriatic response. Isoprenaline may therefore be a candidate substance for enhancing mydriasis when using intracameral mydriatics.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was funded by grants from the KMA Fund, Synfrämjandets Research Fund and Stiftelsen JC Kempes Minnes Stipendiefond.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References