Effect on Diurnal Intraocular Pressure Variation of Eliminating the α-2 Adrenergic Receptor Subtypes in the Mouse

Abstract

purpose. To investigate the effect on circadian variation of intraocular pressure (IOP) of eliminating the α2A-, α2B-, or the α2C-adrenergic receptor subtypes in the mouse.

methods. A microneedle method was used to measure IOP in knockout mice lacking the α2A-, α2B-, or the α2C-receptor (α2A-R^−/−, α2B-R^−/−, α2C-R^−/−), in wild-type mice of the α2B knockout strain (α2B-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), α2A-R^−/− (n = 10), α2B-R^−/− (n = 8), α2B-R^+/+ (n = 8), and α2C-R^−/− (n = 10). In parallel experiments, eyes from the α2A-R^−/−, α2B-R^−/−, α2C-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, α2A-R^−/−, α2B-R^−/−, α2B-R^+/+, and α2C-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, α2B-R^−/−, α2B-R^+/+, and α2C-R^−/−: P < 0.01; α2A-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 α2A-R^−/−, α2B-R^−/−,α2C-R^−/−, or C57BL/6 mice.

conclusions. These results indicate that IOP magnitude and circadian variation are minimally altered by the absence of the α2A-, α2B-, or α2C-receptor subtypes in transgenic mice.

The α2-adrenoreceptors are part of a larger family of adrenergic receptors (including α1- and β2-receptors) that respond to norepinephrine released by the sympathetic innervation of the eye. Agonists that specifically activate α2-receptors can lower intraocular pressure (IOP) without many of the side effects of sympathomimetics that also activate the other adrenoreceptors. ¹ Blockade of α2-adrenoreceptors with the specific α2-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 α2-agonist brimonidine was significantly less at night than during the day. ³ These observations raise the possibility that α2-adrenoreceptor activation contributes to the normal 24-hour fluctuation of IOP. 

Three subtypes of human α2-adrenergic receptor have been identified and cloned: the α2A-, the α2B-, and the α2C-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 α2-agonists. ^{6 7 8 9 10} Physiological and knockout mouse studies have shown that the α2A-receptor is found predominantly in central nervous system (CNS) presynaptic terminals, the α2B-receptor is found predominantly in CNS postsynaptic terminals, and the α2C-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 α2A-receptor is most abundant and is found in choroid and retina. ^{14 15} The α2C-receptor also may be present in the porcine retina. ¹⁵ In the human anterior segment, the α2B- and the α2C-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 α2A-receptor. ¹⁷ However, the specific functions of the various α2-receptor subtypes in the normal control of IOP throughout the day remain unknown. 

Recently, it has been shown that the anatomy of the anterior segment ¹⁸ 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 α2A-, α2B-, or α2C-receptor. ^{6 12 13} Thus, the present study was undertaken to evaluate the potential role of α2-adrenoreceptors in normal IOP control by assessing whether IOP measured during the day and at night becomes altered in mice lacking the α2A-, α2B-, and α2C-receptor. 

Methods

Animal Husbandry

All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Compared mouse strains included: the C57BL/6 mouse as a background control, an α2A-adrenergic receptor–null mutant mouse (B6.129S2-Adra2atm1Lel), an α2B-adrenergic receptor–knockout mouse (B6.129S2-Adra2btmGsb), an α2B-adrenergic receptor wild-type mouse, and an α2C-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), α2A-receptor (R)^−/− (n = 10), α2B-R^−/− (n = 8), α2B-R^+/+ (n = 8), and α2C-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). 

Results

Evaluation of Early Anesthesia Effects

The SP and DP of the awake mice were 101 ± 8 and 74 ± 5 mm Hg, respectively. Moreover, there was no significant difference among awake BP measurements and BP measurements collected at 4, 6, and 8 minutes after administration of anesthesia (Fig. 1 , n = 10 mice). Overall, mouse IOP measured during this 4- to 8-minute period varied by less than 2% of peak IOP. These results indicate that anesthesia with ketamine-xylazine had little effect on BP during the first 8 minutes. After 8 minutes, BP varied among the tested mice (i.e., in some cases it remained stable, whereas in other cases there was a gradual reduction). IOP measurements between 3 and 8 minutes after anesthesia injection were also stable. Hence, for subsequent measurements of one eye or both eyes, the IOP was recorded within the period of 4 to 8 minutes after anesthesia administration. 

The time course of IOP after anesthesia obtained with ketamine-xylazine or ketamine alone is shown in Figure 2 . Between 5 and 8 minutes after anesthetic injection, there were negligible changes in IOP with ketamine+xylazine or ketamine alone. After this period, there were similar time-dependent declines in IOP for each anesthetic group. This decline in IOP was significant during the 20-minute time period analyzed for each anesthetic (P < 0.0001). The mean IOP was ∼1.3 mm Hg less in the eyes of animals that received ketamine plus xylazine. However, this difference was nonsignificant (P = 0.253). 

Diurnal IOP of Normal and α2-Adrenergic Receptor Knockout Mice

The IOP of C57BL/6 (n = 8), α2A-R^−/− (n = 10), α2B-R^−/− (n = 8), α2B-R^+/+ (n = 8), and α2C-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 . 

Nocturnal IOP of Normal and α2-Adrenergic Receptor-Knockout Mice

Nocturnal IOP of C57BL/6, α2A-R^−/−, α2B-R^−/−, α2B-R^+/+, and α2C-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 α2-Adrenergic Receptor-Knockout Mice

Nocturnal IOP of C57BL/6, α2A-R^−/−, α2B-R^−/−, α2B-R^+/+, and α2C-R^−/− mice was significantly higher than the diurnal IOP measured in the same eye (C57BL/6, α2B-R^−/−, α2B-R^+/+, and α2C-R^−/−: P < 0.01; and α2A-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 α2A-, α2B, or α2C-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. 

Discussion

The present study has shown that IOP measured at 0900 hours in mice lacking the α2A-, α2B-, or α2C-adrenoreceptor is similar to normal C57BL/6 mice and control wild-type α2B-R^+/+ mice. The results indicate that the maintenance of IOP during the day is not critically dependent on signaling by these α2-adrenoreceptor subtypes. Twelve hours later, significant increases in IOP were observed in the normal C57BL/6 mice and wild-type α2B-R^+/+ mice as well as in mice lacking the α2A-, α2B-, or α2C-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 α2A-, α2B, or α2C-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 α2C-R^−/− mice than in α2A-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 α2-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 α2-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, α2-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 α2-adrenergic receptors, inhibition of α1-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, α2A-R^−/−, α2B-R^−/−, and α2C-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 α2-agonist xylazine in the anesthesia cocktail may have altered the IOP readings from the various α2-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 α2-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 α1-antagonists, affect IOP in these mice may provide further insight into the contributions of the α2-adrenoreceptor subtypes to IOP regulation. 

The relevance of these conclusions to humans is dependent in part on the similarity of the mouse and human α2-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 α2A receptors in mice, rats, and certain other species that results in less sensitivity to the α2-antagonist plant alkaloid yohimbine has led to their designation as α2D-receptors. ⁷ Later studies have shown that overall the amino acid sequences of the α2A and α2D 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 α2A/D or α2A 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 α2-receptor subtype distribution in rabbit and human eye found both differences as well as similarities in the expression of the three α2-receptor subtypes. ¹⁶ Thus, further experiments addressing the role of the three α2-receptor subtypes in humans may be helpful. 

In conclusion, the present study has shown that no single α2 receptor is critical in the regulation of diurnal IOP or in mediating the nighttime IOP increase in transgenic mice. 

 Supported in part by National Eye Institute Grant EY05990 (RNW), and the Margaret and Robert Boemer Glaucoma Research Fund of the Foundation for Eye Research (MA).

 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.

 

Figure 1.

 

Comparison of SP (○), 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.

Figure 2.

 

Twenty-minute time course of IOP after anesthesia obtained with ketamine+xylazine or ketamine alone (n = 5 in each group).

Table 1.

 

Diurnal and Nocturnal IOP of α2-Adrenergic Receptor Knockout Mice

Genotype (Short Name)	C57BL/6	α2A-R−/−	α2B-R−/−	α2B-R+/+	α2C-R−/−	ANOVA (P)**
Mice (n)	8	10	8	8	10
IOP at 9:00*	17.1 ± 1.8	17.7 ± 1.4	17.1 ± 2.1	17.6 ± 1.3	17.3 ± 0.9	0.89 (NS)
IOP at 21:00	19.6 ± 1.9	19.2 ± 2.2	20.5 ± 1.4	19.7 ± 0.8	21.3 ± 2.7	0.15 (NS)
Paired t-test 900 h vs. 2100 h (P)	<0.01	<0.05	<0.01	<0.01	<0.01
IOP Change (2100–900 h)	2.5 ± 1.6	1.5 ± 1.6	3.4 ± 1.6	2.2 ± 1.2	4.0 ± 3.1	0.06 (NS)

 Data are expressed as the mean mm Hg ± SD.

Figure 3.

 

IOP observed at 0900 (A), at 2100 (B) hours, and the change in IOP between these two time points (C) in control mice and mice lacking an adrenoreceptor subtype. Control mice examined included the C57BL/6 background strain (Cont.) and the wild-type littermates of the α2B-R^−/− mice (α2B^+/+). The small dots are individual data points. The center horizontal lines within the diamonds indicate the group means. The top and bottom points of the diamonds indicate the 95% confidence interval. The short horizontal lines connected by a vertical line indicate the SD. The horizontal line stretching across the plot indicates no change in IOP (Δ = 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 Table 1 .

Figure 4.

 

Similar organization of anterior segment structures in α2A-R^−/− (A), α2B-R^−/− (B), α2C-R^−/− (C), and C57BL/6 wild-type (D) mice. C, cornea; I, iris; CP, ciliary process; R, retina. Magnification, ×220.

The authors thank Christopher Bowd (University of California San Diego) for assistance in the statistical analysis of the IOP time course experiment.