Role of α₁-Adrenoceptor Subtypes in Pupil Dilation Studied With Gene-Targeted Mice

Abstract

Purpose.: The α_1A-adrenoceptor (α_1A-AR) subtype was suggested to mediate contraction and trophic effects in the iris dilator muscle, and thus its pharmacological blockade may be involved in intraoperative floppy iris syndrome. We tested the hypothesis that the α_1A-AR mediates pupil dilation and trophic effects in the mouse iris.

Methods.: The α₁-AR subtype mRNA expression was quantified in iris tissue by real-time PCR. To assess the role of individual α₁-ARs for mediating pupil dilation, the α₁-AR agonist phenylephrine was topically applied to the ocular surface of mice deficient in one of the three α₁-AR subtypes (α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, respectively) and wild-type controls. Changes in pupil diameter were measured under a microscope in restrained mice. Moreover, iris and iris muscle thickness were determined in cryosections.

Results.: Messenger RNA for all three α₁-AR subtypes was detected the iris of wild-type mice with a rank order of abundance of α_1A ≥ α_1B > > α_1D. The lack of a single α₁-AR gene did not affect mRNA expression of the remaining two receptor subtypes. Phenylephrine induced pupil dilation in wild-type mice that was reduced in extent and duration in α_1A-AR^−/− and, less so, in α_1B-AR^−/− but not in α_1D-AR^−/− mice. The lack of a single α₁-AR subtype had no effect on iris or iris muscle thickness.

Conclusions.: The α₁-AR–induced mydriasis in mice is mediated mainly by the α_1A-AR, with a smaller contribution of the α_1B-AR, matching the relative abundance of these subtypes at the mRNA level. The lack of a single α₁-AR subtype does not appear to cause atrophy in the mouse iris.

Introduction

The α₁-adrenoceptors (α₁-ARs) contribute to various physiological actions in and around the eye, and are, for example, involved in regulation of protein secretion in the lacrimal gland,¹ tone of ocular blood vessels,^2,3 and pupil diameter.⁴ 

The α₁-AR family is composed of three subtypes, denoted α_1A, α_1B, and α_1D.⁵ All three receptor subtypes are activated by catecholamines and can mediate constriction of smooth muscle cells.⁶ Pharmacological studies in various species that used selective antagonists for individual α₁-AR subtypes suggested that the α_1A-AR plays a major role in adrenergic pupil size regulation.^7–12 Clinical support for these findings came in 2005 with the description of “intraoperative floppy iris syndrome” (IFIS), a triad of billowing iris, iris prolapse, and progressive pupil constriction during cataract surgery.¹³ Intraoperative floppy iris syndrome has initially been observed in patients under medication with the α_1A-AR–selective antagonist tamsulosin for treating lower urinary tract symptoms suggestive of benign prostatic hyperplasia,¹³ but meanwhile also has been demonstrated with many other drugs having affinity for α₁-ARs.¹⁴ Although the basis of IFIS is thought to be antagonism of the α_1A-AR subtype located in the iris dilator muscle, there are still some doubts regarding the pathophysiology. One observation that cannot be explained pharmacologically is that IFIS has been reported even years after discontinuation of the medication.^13,15,16 Thus, it has been proposed that IFIS may result from a combination of pharmacologic inhibition of iris smooth muscle contraction and long-term smooth muscle atrophy related to drug accumulation in adjacent iris pigment epithelial cells.¹⁷ 

The goal of the present study was to examine the hypothesis that the α_1A-AR subtype mediates pupil dilation and trophic effects in the mouse iris. Moreover, the contribution of the other two α₁-AR subtypes to these effects has been tested. Because of the lack of highly selective pharmacological blockers for some of the α₁-ARs, gene-targeted mice lacking one of the three α₁-AR subtypes (α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, respectively) were used in this study. 

Materials and Methods

Animals

All studies were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local government. The generation of α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice has been described previously.^18–20 Each genotype has been backcrossed with C57BL/6Slc mice for nine generations and maintained on a C57BL/6Slc background. The genotype of each mouse was determined by PCR of DNA isolated from tail biopsies. Mice were housed under standardized conditions with a 12-hour light/dark cycle, temperature of 22 ± 2°C, humidity of 55% ± 10%, and with free access to food and tap water. For experiments, male mice at the age of 8 to 9 months were used. 

Real-Time PCR Analysis

Expression of α₁-AR mRNA was quantified in iris tissue from wild-type (C57BL/6Slc), α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice using real-time PCR. After mice had been killed by CO₂ inhalation, the eyes were immediately removed and placed in ice-cold PBS (Invitrogen, Karlsruhe, Germany). Then, the iris was isolated by the use of fine-point tweezers under a dissecting microscope, transferred into a 1.5-mL tube, and immediately snap frozen. 

Subsequently, the tissue was homogenized in lysis buffer using a homogenization device (Schwingmühle MM 300; Retsch GmbH, Haan, Germany; Lysing Matrix D MP; MP Biomedicals, Illkirch, France). After homogenization, total RNA was extracted with the RNeasy Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. After isolation, mRNA was reverse transcribed with Moloney Murine Leukemia Virus reverse transcriptase and random hexamers (Promega, Mannheim, Germany). Quantitative PCR analysis was performed with the ViiA 7 system (Applied Biosystems, Darmstadt, Germany). We used SYBR green for the fluorescent detection of DNA generated during PCR. The PCR was performed in a total volume of 10 μL and 2× SYBR Green master mix (QIAGEN); 2 μL cDNA corresponding to 13 ng RNA was used as template. Published sequences for mouse α_1A-AR (NM_013461), α_1B-AR (NM_007416), and α_1D-AR (NM_013460) were used to design primers for PCR amplification. Primer sequences were α_1A-AR sense 5′-GCG GTG GAC GTC TTA TGC T-3′ and antisense 5′-TCA CAC CAA TGT ATC GGT CGA-3′; α_1B-AR sense 5′-CCT GGT CAT GTA CTG CCG A-3′ and antisense 5′-GAC TCC CGC CTC CAG ATT C-3′; α_1D-AR sense 5′-AGT TGG TGA CCG TCT GCA AGT-3′ and antisense 5′-CGC TGT GGT GGG AAC CGG CAG-3′; β-actin sense 5′-CAC CCG CGA GCA CAG CTT CTT T-3′ and antisense 5′-AAT ACA GCC CGG GGA GCA TC-3′. Standard negative controls were used for the PCR. A control lacking reverse transcriptase was used to check for DNA contamination of the isolated RNA. We also used a reverse transcription control without RNA to check for contamination of the chemicals used. In both controls, no PCR product was detected, indicating that genomic DNA was absent and the purity of the chemicals was high. For positive control, we used RNA isolated from mouse brain. The expression levels of individual α₁-AR subtype mRNA were normalized to β-actin using the ΔCt method. Parallelism of standard curves was confirmed. 

Measurement of Pupil Diameter

Pupil diameter was determined in conscious restrained mice under a stereomicroscope (SZ61; Olympus Deutschland GmbH, Hamburg, Germany). First, a photograph of the right eye was taken before the application of eyedrops. The diameter of the pupil in this photograph was considered as the baseline diameter. Then, 4 μL of 5% phenylephrine hydrochloride eyedrops (Neosynephrin-POS 5%; Ursapharm Arzneimittel GmbH, Saarbrücken, Germany) were applied into the right eye by using a micropipette. After 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 minutes, photographs of the right eye were taken. The illumination (≈1000 lux) was standardized and measured before the start of each experiment using a luxmeter (LM37; Carl Roth GmbH, Karlsruhe, Germany). From each photograph, the vertical and horizontal pupil diameters were measured by a masked evaluator and the average calculated. 

Measurement of Iris and Iris Muscle Thickness

For histological examinations, mice were killed and their eyes immediately enucleated. After enucleation, a high interindividual variability in pupil diameter was observed. Although some pupils remained stable for a few minutes, others markedly dilated directly after enucleation. To enable iris and iris muscle measurements under standardized conditions, each eye was placed for 5 minutes into PBS containing 0.03% of carbachol (Isopto-Carbachol 3%; Alcon Pharma GmbH, Freiburg, Germany) at room temperature to constrict the pupil. Next, each eye was embedded in Tissue-Tek O.C.T Compound (Sakura Finetek Germany GmbH, Staufen, Germany) and snap frozen in liquid nitrogen in a sagittal orientation. Frozen tissue samples were sectioned at 10-μm thickness and adhered onto chromium-gelatin–coated slides to prevent tissue detachment. To enable iris and iris muscle measurement at a standardized localization, only central sagittal sections of the eye containing the pupil and the optic nerve head were used for analysis (Fig. 1A). Because the iris sphincter and dilator muscles were partially masked by melanin pigment granula, preventing exact measurement of their thickness, we bleached the tissue sections before examination (Figs. 1B, 1C). For bleaching, we used a protocol that has recently been established by our laboratory to rapidly and effectively remove melanin pigment.²¹ Briefly, cryosections were post-fixed in 4% paraformaldehyde for 20 minutes before depigmentation using 10% H₂O₂ diluted in PBS at 65°C for 120 minutes and routinely stained with hematoxylin and eosin. Iris thickness was not affected by bleaching (data not shown). The iris and sphincter thicknesses of the stained sections were measured at ×200 magnification, whereas the iris dilator thickness was measured at ×400 magnification under a bright-field microscope. For each eye, a total of 12 iris or iris muscle thickness measurements were obtained from standardized positions; 6 from the central and 6 from the peripheral iris region measured in the peripheral, middle, and proximal aspects, respectively. 

Figure 1

 

Sagittal cryosection of a murine eye globe (A). For evaluation of iris and iris muscle thickness, central sections containing the pupil and optic nerve head were used to enable standardized measurements. The arrowheads point to the central iris region containing the iris sphincter muscle. The region between the central iris region and the ciliary body (thin arrows) is the peripheral iris region containing the dilator muscle. The thick arrow points to the optic nerve head. Central and peripheral iris thicknesses, as well as iris sphincter (B) and dilator (C) thicknesses, were measured at six different sites per eye, respectively. The arrows in (B) and (C) point to the borders of the muscles. a, anterior; p, posterior.

Statistical Analysis

Data of mRNA expression are presented as box plots with the ends of the whiskers representing the minimum and maximum. For statistical analysis of α₁-AR mRNA expression levels, the Kruskal-Wallis test, followed by the Dunn's multiple comparison test, was used. Pupil responses are presented as percentage of change in diameter from baseline diameter (mean ± SE), and for comparisons of time-dependent pupil responses between individual mouse genotypes, repeated-measures ANOVA followed by the Tukey test were used. Iris and iris muscle thicknesses were expressed as mean ± SE and compared by one-way ANOVA followed by the Tukey test. The level of significance α was set at 0.05. Multiple comparisons were tested at a Bonferroni-adjusted α level, and n represents the number of mice per group 

Results

Expression of α₁-AR in the Iris

Using real-time PCR, we detected mRNA of all three α₁-AR subtypes in iris tissue from wild-type mice (Fig. 2). Remarkably, α_1A-AR mRNA and α_1B-AR mRNA were more abundant than α_1D-AR mRNA. No significant difference was seen between α_1A-AR mRNA and α_1B-AR mRNA expression levels. In mice lacking a single α₁-AR subtype, no compensatory changes in mRNA expression for the remaining two subtypes were observed (Fig. 3). 

Figure 2

 

Relative mRNA expression of individual α₁-AR subtypes normalized to β-actin transcripts in iris tissue from wild-type mice. Data are presented as box plots with the ends of the whiskers representing the minimum and maximum (n = 7; ***P < 0.001 α_1A versus α_1D; *P < 0.05 α_1B versus α_1D).

Figure 3

 

The mRNA expression levels for the α_1A-AR (A), α_1B-AR (B), and α_1D-AR subtypes (C) were compared in iris tissue from individual mouse genotypes. Expression levels are normalized to β-actin transcripts and the data are expressed as box plots with the ends of the whiskers representing the minimum and maximum (n = 7–9 per genotype).

Pupil Diameter Changes in Response to Phenylephrine

Baseline pupil diameter before application of phenylephrine was 566 ± 25 μm, 587 ± 19 μm, 564 ± 21 μm, and 553 ± 26 μm in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively, and did not differ between individual mouse genotypes. In wild-type mice, phenylephrine evoked marked pupil dilation already 2 minutes after application (Fig. 4). Change in pupil diameter from baseline reached its maximum of 49% ± 5% 4 minutes after phenylephrine application and remained constant for 20 minutes. In α_1D-AR^−/− mice, similar to wild-type mice, a maximum of 51% ± 10% in pupil dilation from baseline was observed after 4 minutes, and the mydriasis also remained stable for 20 minutes. In α_1B-AR^−/− mice, the maximum change in pupil diameter was 39% ± 8% and slightly smaller compared with wild-type and α_1D-AR^−/− mice, and the diameter already started to decrease within the time frame of 20 minutes. The time course of pupil dilation differed significantly from that of wild-type and α_1D-AR^−/− mice. In α_1A-AR^−/− mice, the maximal change of pupil diameter in response to phenylephrine was weakest among the four mouse genotypes and only 21% ± 5% from baseline diameter. The pupil diameter also decreased within the time frame of 20 minutes almost to the baseline level. In α_1A-AR^−/− mice, the pupil response to phenylephrine differed significantly compared with wild-type, α_1D-AR^−/−, and α_1B-AR^−/− mice. 

Figure 4

 

Representative pictures of eyes from a wild-type mouse made during pupil diameter measurements before (A) and 4 minutes after application of phenylephrine (B) to demonstrate changes in pupil diameter. (C) Graph showing relative changes in pupil diameter from wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice to the α₁-AR agonist phenylephrine. Values are expressed as mean ± SE (n = 12 per genotype; ***P < 0.001 α_1A versus all other groups; **P < 0.01 α_1B versus wild-type and α_1D).

Iris Morphology

Carbachol-preconstricted pupil diameter assessed in sagittal cryosections of the mouse eye globe was 354 ± 23 μm, 327 ± 24 μm, 348 ± 27 μm, and 365 ± 42 μm, and did not differ between individual mouse genotypes. Central iris thickness was 64 ± 3 μm, 58 ± 2 μm, 59 ± 2 μm, and 66 ± 2 μm in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively, and did not differ among the four mouse genotypes (Fig. 5A). Peripheral iris thickness was 29 ± 1 μm, 28 ± 1 μm, 28 ± 1 μm, and 30 ± 1 μm in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively, and was also similar among all genotypes (Fig. 5B). The thickness of the iris sphincter muscle was 25 ± 1 μm, 23 ± 1 μm, 25 ± 1 μm, and 27 ± 1 μm in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively, and was also similar in the four mouse genotypes (Fig. 5C). Also, the dilator muscle had a similar thickness in all genotypes: 9.1 ± 0.2 μm, 8.7 ± 0.2 μm, 9.0 ± 0.3 μm, and 9.3 ± 0.3 μm in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively (Fig. 5D). 

Figure 5

 

Thickness of central (A) and peripheral iris (B) and of iris sphincter (C) and dilator (D) in wild-type (WT), α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice. No differences were found among the four mouse genotypes. Values are expressed as mean ± SE (n = 10–11 per genotype).

Discussion

There are several major new findings in the present study. First, pupil responsiveness to adrenergic stimuli in mice was mediated predominantly by the α_1A-AR subtype with a minor contribution of the α_1B-AR subtype. Second, mRNA expression levels of individual α₁-AR subtypes were in good agreement with the contribution of the respective subtypes to pupil dilation, and disruption of a single α₁-AR subtype gene did not have significant effects on the mRNA expression levels of the remaining two receptor subtypes, which is in line with previous studies in other tissues.^20,22 Because the specificity of commercially available antibodies directed against individual α₁-AR subtypes appears to be limited in mice,^23,24 we chose not to quantify receptor proteins. Third, the lack of a single α₁-AR subtype did not affect iris and iris muscle thickness. 

This is the first study in mice that has investigated the expression of individual α₁-AR subtypes in the iris and their involvement in adrenergic pupil diameter regulation. Previous studies in rats²⁵ and rabbits^9,12,26 have demonstrated that in iris tissue the α_1A-AR subtype is either expressed most abundantly or equally as high as the α_1B-AR, whereas the α_1D-AR subtype is least abundantly expressed both at the mRNA and protein levels. Our study extends these findings to the mouse iris, indicating that this is well conserved in mammals. 

The functional role of α₁-ARs has been examined in various animal species. For example, in cats, the receptor mediating iris dilation has been described as an atypical receptor, unclassifiable as either α₁-AR or α₂-AR.²⁷ Studies in rats suggested that sympathetic mydriasis is mediated by the α_1A-AR subtype.^7,8 However, it has been suggested that the α_1B-AR may be involved in mediating contraction of rat irideal blood vessels.²⁸ An attempt to assess the functional relevant α₁-AR subtype in pigs has been hampered, because binding of the α₁-AR antagonist prazosin to melanin was too high to detect any of the α₁-AR subtypes.¹² Many functional studies have been performed in rabbits. Most of them indicated that α_1A-AR is the main mediator of adrenergic pupil dilation, but differences in affinity to prazosin have been observed dependent on the state of iris pigmentation.^4,9,11,29 Another study suggested that the low-affinity phenotype of the α_1A-AR mediates adrenergic pupil dilation in humans.⁴ The present study in mice is in agreement with most of the previous studies conducted in other species, demonstrating that the α_1A-AR subtype plays a predominant role in adrenergic pupil dilation. 

Another intriguing question addressed in the present study was whether the lack of individual α₁-AR subtypes resulted in iris dilator thinning. Because IFIS has been observed in patients who had been off tamsulosin for more than 1 year, Chang and Campbell¹³ hypothesized that a “disuse atrophy” of the iris dilator muscle caused by chronic pharmacological blockade of α₁-ARs may contribute to the syndrome. Studies in humans who had been on medication with α₁-AR antagonists, primarily tamsulosin, supported this hypothesis by demonstrating peripheral iris or iris dilator muscle thinning.^30,31 Moreover, a case series in humans on topical medication with the α₁-AR antagonist, bunazosin, reported signs of iris dilator muscle atrophy together with changes of pigment granules in melanocytes and clump cells, and thus suggested that IFIS should not only be attributed to binding of a specific drug to α₁-ARs, but also to a drug-melanin interaction causing dilator muscle atrophy.³² In support of this concept, a recent study in rabbits suggested that chronic exposure to the α₁-AR antagonists tamsulosin and silodosin causes atrophy of the iris dilator muscle due to a drug-melanin interaction in adjacent pigment epithelial cells.¹⁷ 

In the present study, we found no differences in iris dilator muscle thickness between wild-type mice and any of the mouse genotypes lacking a single α₁-AR subtype. There are several possible explanations for the differences between our observations in mice and the previous in humans and rabbits. 

First, species differences are a possible factor that needs to be considered. Although in humans and rabbits, like in mice, adrenergic pupil dilation appears to be mediated predominantly by the α_1A-AR subtype, we cannot rule out the possibility of species differences in the coupling of α₁-ARs to growth responses. For example, a study in cultured rat cardiomyocytes that used different α₁-AR antagonists suggested that the α_1A-AR subtype mediates trophic effects in these cells.³³ In contrast, studies in α₁-AR knockout mice demonstrated that the α_1B-AR subtype exerted trophic effects in response to α₁-AR agonists in the heart and in blood vessels.^34,35 Although the lack of the α_1B-AR subtype could not be compensated in the cardiovascular system by the remaining two subtypes in this knockout mouse model, we cannot completely exclude the possibility that in the iris of knockout mice a functional compensation by the remaining two α₁-AR subtypes or other receptors may have occurred so that potential α₁-AR–mediated trophic effects were eventually masked. Second, a thinning of the dilator muscle or the peripheral iris in α₁-AR antagonist-treated subjects found in previous studies may have been secondary to reduced pupil diameter and thus increased stretch and thinning of the peripheral iris. In contrast to the present study, in which iris and iris muscle thickness were measured in histological sections with a standardized pupil diameter, the pupil diameter was either not reported or smaller in α₁-AR antagonist-treated subjects in the previous studies.^17,30,31 Third, all previous studies measured iris and iris muscle thickness in subjects treated with α₁-AR antagonists, whereas in the present study, the measurements were performed in mice lacking single α₁-AR subtypes. Hence, it is also possible that in the previous studies another factor, not necessarily related to an α₁-AR signaling pathway, caused iris dilator atrophy due to chronic exposure to the respective antagonists. A drug-melanin interaction, as suggested by Goseki et al.,^17,32 may be one possible explanation. Because the interaction between all α₁-AR antagonists and the receptors is reversible and because there are no data from other tissues indicating that blockade of α₁-ARs causes irreversible atrophy, there remains the unresolved question as to why IFIS can occur even years after drug discontinuation. 

In conclusion, this is the first study to provide evidence that α_1A-AR and α_1B-AR subtypes are most abundantly expressed and functionally active in the mouse iris. The mRNA expression data and the pharmacological findings are in line with previous studies in other species, including humans. However, we found no evidence that the lack of a single α₁-AR subtype causes atrophy in the mouse iris. The α₁-AR knockout mice may serve as a model to examine the effects of drugs causing IFIS. 

Acknowledgments

We thank Paul C. Simpson (Cardiology Section, San Francisco Veterans Affairs Medical Center, and Department of Medicine, Cardiology Division, University of California, San Francisco, CA, USA), Susanna Cotecchia (Department of Biosciences, Biotechnology and Biopharmaceutis, University of Bari, Italy, and Department of Pharmacology and Toxicology, University of Lausanne, Switzerland), Akito Tanoue (Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan), and Atsushi Sanbe (Department of Pharmacotherapeutics, School of Pharmacy, Iwate Medical University, Iwate, Japan) for making α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, and C57BL/6Slc wild-type mice available for this study. We also thank Brigitte Ruhland (Department of Internal Medicine II, University Medical Center Regensburg, Germany) for expert technical assistance with real-time PCR experiments. Data are part of the doctoral dissertation of CA. 

Supported by a grant from the Gertraud Maria Rzehulka Foundation. 

Disclosure: M.L. Kordasz, None; C. Manicam, None; A. Steege, None; E. Goloborodko, None; C. Amato, None; P. Laspas, None; C. Brochhausen, None; N. Pfeiffer, None; A. Gericke, None