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-2 Adrenergic Receptor Subtypes in the Mouse1From the Hamilton Glaucoma Center, University of California San Diego, La Jolla, California; and the 2Department of Ophthalmology, University of Tokyo, Tokyo, Japan.
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2A-, 2B-, or the 2C-adrenergic receptor subtypes in the mouse.
METHODS. A microneedle method was used to measure IOP in knockout mice lacking the 2A-, 2B-, or the 2C-receptor (2A-R–/–, 2B-R–/–, 2C-R–/–), in wild-type mice of the 2B knockout strain (2B-R+/+), and in the background strain mice, C57BL/6. All mice were maintained in a 12-hour light–dark cycle commencing at 0600 hours. IOP was measured at 0900 and 2100 hours in the five groups: C57BL/6 (n = 8), 2A-R–/– (n = 10), 2B-R–/– (n = 8), 2B-R+/+ (n = 8), and 2C-R–/– (n = 10). In parallel experiments, eyes from the 2A-R–/–, 2B-R–/–, 2C-R–/–, and C57BL/6 mice were embedded in epoxy resin, and semithin sections were stained with toluidine blue.
RESULTS. IOP at 0900 hours in B6, 2A-R–/–, 2B-R–/–, 2B-R+/+, and 2C-R–/– mice was 17.1 ± 1.8, 17.7 ± 1.4, 17.1 ± 2.1, 17.6 ± 1.3, and 17.3 ± 0.9 mm Hg, respectively (mean ± SD). IOP at 2100 hours in the same eyes was 19.6 ± 1.9, 19.2 ± 2.2, 20.5 ± 1.5, 19.7 ± 0.8, and 21.3 ± 2.7 mm Hg, respectively. There was no significant difference among these genotypes in IOP measured at either time point (P > 0.05, ANOVA). Within each genotype, IOP at 2100 hours was significantly higher than IOP at 0900 hours (C57BL/6, 2B-R–/–, 2B-R+/+, and 2C-R–/–: P < 0.01; 2A-R–/–: P < 0.05, paired t-test). Differences in the diurnal IOP change among the different genotypes were insignificant (P > 0.05, ANOVA). Histopathologic assessment found minimal differences in the structural organization of the anterior segment among the 2A-R–/–, 2B-R–/–,2C-R–/–, or C57BL/6 mice.
CONCLUSIONS. These results indicate that IOP magnitude and circadian variation are minimally altered by the absence of the 2A-, 2B-, or 2C-receptor subtypes in transgenic mice.
2-adrenoreceptors are part of a larger family of adrenergic receptors (including 1- and β2-receptors) that respond to norepinephrine released by the sympathetic innervation of the eye. Agonists that specifically activate 2-receptors can lower intraocular pressure (IOP) without many of the side effects of sympathomimetics that also activate the other adrenoreceptors.1 Blockade of 2-adrenoreceptors with the specific 2-antagonists yohimbine or rauwolscine reduced IOP in both the light and dark phase.2 Moreover, IOP reduction and associated reduction of ciliary blood flow after twice-daily treatment with the specific 2-agonist brimonidine was significantly less at night than during the day.3 These observations raise the possibility that 2-adrenoreceptor activation contributes to the normal 24-hour fluctuation of IOP.
Three subtypes of human 2-adrenergic receptor have been identified and cloned: the 2A-, the 2B-, and the 2C-receptor.4 5 6 These subtypes contain similarities within their amino-acid sequences but differ in their posttranslational modifications, their tissue locations, and their responses to various 2-agonists.6 7 8 9 10 Physiological and knockout mouse studies have shown that the 2A-receptor is found predominantly in central nervous system (CNS) presynaptic terminals, the 2B-receptor is found predominantly in CNS postsynaptic terminals, and the 2C-receptor is present mainly at peripheral nervous system (PNS) pre- and postsynaptic terminals.5 6 Moreover, these subtypes play different roles in the control of heart rate and blood pressure.11 12 13 In human and porcine eyes, the 2A-receptor is most abundant and is found in choroid and retina.14 15 The 2C-receptor also may be present in the porcine retina.15 In the human anterior segment, the 2B- and the 2C-receptors are present in the non-pigmented epithelium and the ciliary muscle.16 Moreover, porcine ciliary artery contraction induced by brimonidine is mediated by the 2A-receptor.17 However, the specific functions of the various 2-receptor subtypes in the normal control of IOP throughout the day remain unknown.
Recently, it has been shown that the anatomy of the anterior segment18 and aqueous outflow pathways,19 the IOP,20 21 22 and the circadian rhythm of IOP are similar in mice,23 humans,24 and rabbits.25 26 In addition, knockout mouse strains have been developed that lack the 2A-, 2B-, or 2C-receptor.6 12 13 Thus, the present study was undertaken to evaluate the potential role of 2-adrenoreceptors in normal IOP control by assessing whether IOP measured during the day and at night becomes altered in mice lacking the 2A-, 2B-, and 2C-receptor.
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2A-adrenergic receptor–null mutant mouse (B6.129S2-Adra2atm1Lel), an 2B-adrenergic receptor–knockout mouse (B6.129S2-Adra2btmGsb), an 2B-adrenergic receptor wild-type mouse, and an 2C-adrenergic receptor–knockout mouse (B6.129S2-Adra2ctm1Gsb). As a background strain control, C57BL/6 mice were obtained from Harlan Sprague-Dawley (San Diego, CA). The remaining mouse strains were developed in the laboratory of Brian Kobilka (Stanford University, Palo Alto, CA). The specific ablation of each of these receptor subtypes in these mice has been well characterized in previous studies.12 13 All mice were housed in clear plastic cages covered loosely with air filters and containing white pine shavings for bedding. Feeding was ad libitum and room temperature was 21°C. All mice were exposed to a 12-hour light (0600–1800 hours)–dark (1800–0600 hours) cycle for at least 2 weeks before IOP was measured.
Measurement of IOP
The mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (9 mg/kg, TranquiVed; VEDCO Inc., St. Joseph, MO). Intraocular pressure was directly measured in both eyes within the first 7 minutes after injection of anesthesia by a microneedle method described previously.21 A 2-week recovery period separated subsequent IOP measurements on the same eye.
Evaluation of Early Anesthesia Effects
To determine the time course of anesthesia’s effects on blood pressure (BP) and IOP, systolic (SP) and diastolic (DP) blood pressure and IOP were simultaneously measured in 11 NIH Swiss white mice. A noninvasive blood pressure system (Kent Scientific, Torrington, CT) was used that balanced constriction of blood flow in the mouse tail with an inflatable cuff against the output of a sensitive plethysmographic cuff placed adjacent to the blood flow cuff. Pressure in the blood flow cuff was varied by microprocessor control and measurements were collected automatically. For each experiment, the mouse was acclimated to a plastic restrainer for 30 minutes. Then, awake SP and DP were measured four times. Next, ketamine-xylazine anesthesia was injected, and a timer was started. After it lost consciousness, the mouse was removed from the restrainer without disturbing the tail cuffs and positioned for microneedle IOP measurement. Loss of response to tail-pinch stimulation was confirmed at 3 minutes after anesthetic injection. Then, SP, DP, and IOP were measured simultaneously over the course of the next 5 minutes. Briefly, the microneedle was inserted in one eye and left in place. IOP was recorded every 15 seconds. Each measurement of SP and DP required 30 seconds. Hence, measurements were collected during the 30 seconds before and 4, 6, and 8 minutes after injection. Each of these measurements was immediately repeated. The two measurements collected immediately before and after the 4-, 6-, or 8-minute time points were then averaged.
To investigate whether the presence of xylazine altered the measurement of IOP, the time course of IOP was assessed during anesthesia obtained with either ketamine-xylazine as just described or during anesthesia obtained with injection of ketamine alone (150 mg/kg, IP). Anesthesia adequate for insertion of the microneedle usually was obtained by 4.0 to 4.5 minutes after injection. Measurements of IOP were then recorded at 1-minute intervals between 5 and 20 minutes after anesthesia injection.
Diurnal and Nocturnal IOP measurement
Diurnal and nocturnal IOP was measured at 900 and 2100 hours in five groups: C57BL/6 (n = 8), 2A-receptor (R)–/– (n = 10), 2B-R–/– (n = 8), 2B-R+/+ (n = 8), and 2C-R–/– (n = 10). One eye in each mouse was selected randomly for analysis. Until the mouse was placed on the table for IOP measurement, room lighting was similar to that in the vivarium room. During the IOP measurement, the mouse eye was evenly illuminated with a fiber optic illuminator. IOP was measured within 2 minutes after turning on this light. For measurements obtained during the dark phase, the animals were kept in the room with the IOP measurement equipment to minimize handling before anesthesia, and injection of anesthesia was performed under dim light illumination to minimize alteration of IOP by light perception.22
Histopathologic Assessment
Deeply anesthetized mice were exsanguinated by transcardial perfusion with mammalian Ringer’s solution and then fixed by transcardial perfusion with 2% glutaraldehyde and 2% paraformaldehyde in 0.15 M cacodylate buffer (pH 7.3). After enucleation, fixation continued in the perfusion fixative for 1 hour. The eyes were then postfixed in 1.0% osmium tetroxide in cacodylate buffer for 1 hour, dehydrated through graded ethanols and acetone, and embedded in Epon araldite (Electron Microscopy Sciences, Hatfield, PA). Sections 1 µm thick were produced by glass knives on an ultramicrotome and then stained with toluidine blue.
Statistical Analysis
All data are presented as the mean ± SD. The differences in IOP among five genotypes at each time point were assessed by one-way analysis of variance (ANOVA) and the Student-Newman-Keuls (SNK) t-test. The difference between diurnal and nocturnal IOP for each of the genotypes was assessed by paired t-test. The time courses of IOP after anesthesia with either ketamine plus xylazine or ketamine alone were compared by using a mixed-model ANOVA with one within-subject factor (time) and one between-subjects factor (anesthesia type, with two levels).
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1.3 mm Hg less in the eyes of animals that received ketamine plus xylazine. However, this difference was nonsignificant (P = 0.253).
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2-Adrenergic Receptor Knockout Mice2A-R–/– (n = 10), 2B-R–/– (n = 8), 2B-R+/+ (n = 8), and 2C-R–/– (n = 10) mice measured at 0900 hours was 17.1 ± 1.8, 17.7 ± 1.4, 17.1 ± 2.1, 17.6 ± 1.3, and 17.3 ± 0.9 mm Hg, respectively. There was no significant difference in these genotypes (P > 0.05, ANOVA; Table 1 ). This result is consistent with the overlap of the 95% confidence intervals for each genotype shown in Figure 3A .
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2-Adrenergic Receptor-Knockout Mice2A-R–/–, 2B-R–/–, 2B-R+/+, and 2C-R–/– mice measured at 2100 hours in the same eye was 19.6 ± 1.9, 19.2 ± 2.2, 20.5 ± 1.5, 19.7 ± 0.8, and 21.3 ± 2.7 mm Hg, respectively. There was no significant difference in these genotypes (P > 0.05, ANOVA; Table 1 ). This result is consistent with the overlap of the 95% confidence intervals for each genotype shown in Figure 3B .
Diurnal Change of IOP in Normal and 2-Adrenergic Receptor-Knockout Mice
Nocturnal IOP of C57BL/6, 2A-R–/–, 2B-R–/–, 2B-R+/+, and 2C-R–/– mice was significantly higher than the diurnal IOP measured in the same eye (C57BL/6, 2B-R–/–, 2B-R+/+, and 2C-R–/–: P < 0.01; and 2A-R–/–: P < 0.05; paired t-test, Table 1 ). Comparison of the diurnal IOP change among the different genotypes by ANOVA showed that the variations in these differences were nonsignificant. As shown in Figure 3C , the 95% confidence interval for each genotype did not overlap 0 mm Hg (no change in IOP) but did overlap the 95% confidence interval of IOP change for each of the other genotypes. This result is consistent with a significant IOP increase at night in each genotype and the absence of a significant difference between the various genotypes.
Histopathologic Assessment
The structure of the anterior segment tissues of the mice lacking the 2A-, 2B, or 2C-adrenoreceptor as well as the C57BL/6 mice was evaluated in 1-µm-thick plastic sections. As shown in Figure 4 , there was similar organization of anterior segment tissues, including the ciliary body, trabecular meshwork, and sclera in the various strains. These results suggest that there were no significant structural differences in these knockout mouse groups.
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2A-, 2B-, or 2C-adrenoreceptor is similar to normal C57BL/6 mice and control wild-type 2B-R+/+ mice. The results indicate that the maintenance of IOP during the day is not critically dependent on signaling by these 2-adrenoreceptor subtypes. Twelve hours later, significant increases in IOP were observed in the normal C57BL/6 mice and wild-type 2B-R+/+ mice as well as in mice lacking the 2A-, 2B-, or 2C-adrenoreceptor. As there was no significant difference in either the magnitude of the IOP at 2100 hours or in the change of IOP between 0900 and 2100 hours, it does not appear that the 2A-, 2B, or 2C-adrenoreceptor is critically involved in the nocturnal elevation of IOP in the mouse eye.
It is possible that subtle differences in these parameters exist among these mice that might have been resolved if the number of mice in each group were larger. For example, the mean IOP at 2100 hours was 2.1 mm Hg greater in 2C-R–/– mice than in 2A-R–/– mice. In view of the variability of the measured IOP in these mice, groups with 44 or more mice each would be needed to determine whether this difference was significant. A similarly large number of animals would be needed to determine whether the difference in the change of IOP between these two groups was significant. However, these determinations would not alter the general conclusion that the presence of these 2-receptor subtypes is not required for maintenance of essentially normal IOP during the day and essentially normal IOP elevation at night. To investigate further, we conducted a post hoc power analysis of the results by using the average elevation of IOP at night (2.72 mm Hg), the average number of mice per group,9 the average SD observed (1.82 mm Hg), and a 0.05 level of significance. We observed that these parameters yielded a power of 0.88, a level that strongly supports the statistical conclusions that we obtained.
The current results indicate that no 2-receptor subtype alone is critical for maintenance of IOP during the day or for mediating nocturnal IOP increase. As these mice are transgenic, this conclusion pertains to receptors in the eye as well as in all other parts of the body. There are two possible explanations: First, 2-receptors may be minimally involved in the normal endogenous regulation of IOP. Alternatively, it is possible that the absence of these receptors is readily compensated for by the action of other receptor systems in the eye. This latter possibility is consistent with studies showing that in addition to the IOP changes caused by inhibition of 2-adrenergic receptors, inhibition of 1-adrenergic receptors can reduce nocturnal IOP elevation.2 23 Similarly, inhibition of β2-adrenergic receptors also reduces nocturnal IOP elevation.23 Further studies of mice lacking several of the receptor subtypes may help to clarify this point.
An important consideration is whether variation in resting BP among the various knockout strains could have affected ciliary perfusion or episcleral venous pressure. Previous studies have shown that there is no difference in resting BP among wild-type, 2A-R–/–, 2B-R–/–, and 2C-R–/– mice.12 13 These observations suggest that BP changes did not influence the present daytime IOP measurements significantly. However, it should be noted that in the presence of certain drug treatments or physiological stress, some hemodynamic differences were observed.12 13
Another consideration is whether the use of the 2-agonist xylazine in the anesthesia cocktail may have altered the IOP readings from the various 2-receptor subtype knockout strains. If this were likely, then further studies with other anesthetics lacking xylazine would be needed to determine whether the difference reflects the contribution of that receptor subtype to the observed IOP or the different regulation of IOP. However, no significant differences in the daytime or evening IOP readings were observed. Moreover, no changes were recorded in IOP, SP, or DP during the first 8 minutes after intraperitoneal anesthetic injection. Also, there was no significant difference between IOP obtained with ketamine and xylazine, or ketamine alone during this period. Thus, it appears that loss of consciousness precedes significant changes in either BP or IOP and provides a time window during which invasive measurements reflect awake status. This conclusion supports the view that there was negligible influence of anesthesia on IOP measurements in 2-receptor subtype knockout mice in the present study as these measurements were collected within the first 7 minutes after injection of anesthetic.
It is not known whether there are alterations in the normal circadian fluctuation of BP in these knockout mice. In a recent human study, the investigators found that circadian changes in episcleral venous pressure were independent of BP.27 Hence, direct influence of BP changes on episcleral venous pressure or IOP in these mice may be minimal. Nevertheless, confirming this point, as well as investigating how other IOP-altering agents, such as 1-antagonists, affect IOP in these mice may provide further insight into the contributions of the 2-adrenoreceptor subtypes to IOP regulation.
The relevance of these conclusions to humans is dependent in part on the similarity of the mouse and human 2-adrenoreceptor subtypes. Cloning and sequencing of these subtypes in mice and other species indicate that the amino acid sequence of these receptors is highly conserved. Moreover, the pharmacologic properties of these subtypes are generally similar across species. However, a single amino acid difference within the fifth transmembrane region of 2A receptors in mice, rats, and certain other species that results in less sensitivity to the 2-antagonist plant alkaloid yohimbine has led to their designation as 2D-receptors.7 Later studies have shown that overall the amino acid sequences of the 2A and 2D subtypes are approximately 90% identical and have not been detected together in a single species. Thus, they should be considered species orthologues and may be referred to as the 2A/D or 2A receptors.5 6 8 28 Although these results raise the possibility that the present findings may also pertain to human eyes, molecular, and immunohistochemical comparison of 2-receptor subtype distribution in rabbit and human eye found both differences as well as similarities in the expression of the three 2-receptor subtypes.16 Thus, further experiments addressing the role of the three 2-receptor subtypes in humans may be helpful.
In conclusion, the present study has shown that no single 2 receptor is critical in the regulation of diurnal IOP or in mediating the nighttime IOP increase in transgenic mice.
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Acknowledgements |
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Submitted for publication March 31, 2007; revised June 11 and September 7, 2007; accepted January 7, 2008.
Disclosure: M. Aihara, None; J.D. Lindsey, None; R.N. Weinreb, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Robert N. Weinreb, Hamilton Glaucoma Center, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0946; weinreb{at}eyecenter.ucsd.edu.
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-adrenergic receptors participate in control of the circadian rhythm of IOP?. Invest Ophthalmol Vis Sci. 1992;33:3186–3194.2-adrenergic receptors: comparison of pharmacologically defined subtypes with subtypes identified by molecular cloning. Mol Pharmacol. 1992;42:1–5.[Abstract]2-adrenergic receptor subtype diversity: one receptor is not enough. Am J Physiol. 2002;283:R287–R295.[Web of Science]2-adrenoceptor-subtype function. Trends Pharmacol Sci. 1997;18:211–219.[Medline][Order article via Infotrieve]2-adrenergic receptor subtypes and identification of a single amino acid in the mouse 2-C10 homolog responsible for an interspecies variation in antagonist binding. Mol Pharmacol. 1992;42:16–27.[Abstract](2A/D)-adrenoceptor knockout mice. Br J Pharmacol. 2002;136:857–864.[CrossRef][Web of Science][Medline][Order article via Infotrieve]2 adrenergic receptor subtypes. Ann NY Acad Sci. 1995;763:1–7.[Medline][Order article via Infotrieve]2-adrenoceptors: more subtypes but fewer functional differences. Trends Pharmacol Sci. 1994;15:119–123.[CrossRef][Medline][Order article via Infotrieve]2A-adrenergic receptor subtype. Science. 1996;273:801–803.[Abstract]2-adrenergic receptor subtypes B and C. Science. 1996;273:803–805.[Abstract]2-adrenergic receptor subtype function. Rev Physiol Biochem Pharmacol. 2001;142:161–185.[Web of Science][Medline][Order article via Infotrieve]2 adrenergic receptor subtypes in human ocular tissue homogenates. Invest Ophthalmol Vis Sci. 1999;40:2299–2306.2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies. Invest Ophthalmol Vis Sci. 1995;36:2729–2739.2-adrenoceptor subtypes in the porcine eye: identification of 2A-adrenoceptors in the choroid, ciliary body and iris, and 2A- and 2C-adrenoceptors in the retina. Exp Eye Res. 1996;63:57–66.[CrossRef][Web of Science][Medline][Order article via Infotrieve]2A-adrenoceptor-mediated vasoconstriction by brimonidine in porcine ciliary arteries. Invest Ophthalmol Vis Sci. 2001;42:2049–2055.2-adrenergic receptor genes in rat tissues. Receptor. 1994;4:191–199.[Web of Science][Medline][Order article via Infotrieve]
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), DP (•), and IOP (
) in 10 mice while awake (pre) and after injection of anesthesia (arrow). Time indicates minutes after the injection. The numerical values for the SP and DP measurements are also indicated.
= 0 mm Hg). Note that the 95% confidence interval for each genotype excludes this line, but overlaps the 95% confidence interval for each of the other genotypes. This result is consistent with a significant IOP increase at night within each genotype and the absence of a significant difference among the genotypes shown in 