A broad range of IOPs between strains
Figure shows the average IOP of a number of inbred mouse strains that were housed in the same environmental conditions. There is a wide range of IOP with strain BALB/cJ having one of the lowest average IOPs (11.1 ± 0.5 mmHg) and strain CBA/CaJ one of the highest IOPs (19.3 ± 0.3 mmHg). Significant differences exist among various strains (P < 0.0001 for all groups, ANOVA comparing strains within each sex group).
Figure 1 An almost two-fold range of IOP between genetically distinct mouse strains. Mean IOP ± SEM is shown for each strain. Twelve to 25 mice were analyzed for each strain (typically 18 to 20), except for RIIIS/J (7 mice). Mice of most strains were 6 (more ...)
Clinical and histological analysis of the eyes of all studied strains (see Table ) did not identify anatomic or pathologic features that might account for the differences in IOP. For example, the iridocorneal angle and aqueous humor drainage structures are open to the anterior chamber and have normal morphology in both BALB/cJ and CBA/CaJ mice (Figure ). More than 20% of CBA/CaJ mice had IOPs of over 21 mmHg, which increases risk for glaucoma in humans. We aged a small group of these mice (n = 4) to 2 years and histologically analyzed their optic nerves and retinas but they did not develop glaucoma.
Ocular abnormalities in various strains
Figure 2 Normal iridocorneal angles in strains with high and low IOP. The iridocorneal angle that contains the aqueous humor drainage structures (Schlemm's canal (SC) and trabecular meshwork (TM) had a normal morphology in strains CBA/CaJ (A, high IOP), BALB/cJ (more ...)
Strain differences are reproducible
To assess the consistency of IOP in specific strains, we measured IOP in different cohorts of each strain maintained under similar conditions at different times. We purposefully included strains at each end of the IOP spectrum, and strain C57BL/6J (B6) that is commonly used for genetic experiments (Figure ). The average IOP of different cohorts of strains CBA/CaJ, CBA/CaHN (both high end of spectrum) and B6 were consistent over time. This was true of most strains assessed on multiple occasions. Average IOP for age matched mice of the same strain assessed at different times typically differed by no more than 1.5 mmHg, and the differences were usually smaller. Strain 129P3/J was the most variable strain with the average IOP fluctuating by up to 2.5 mmHg.
Figure 3 The IOPs of different strains are stable and reproducible over time. Mean IOP ± SEM is shown for the indicated strain at different times of measurement. The measurement times within a single year were separated by a few months. The IOPs of strains (more ...)
Despite the general consistency of IOP, the average IOPs of some strains have changed in a reproducible manner. The IOPs of BALB/cJ mice (low end of spectrum) were very similar for the past several years, around 11 mmHg. Between early 1996 and 1997, however, the IOP of this strain did jump from approximately 7.7 mmHg to approximately 11 mmHg (Figure ). During this period, the room in which our animals were housed and the manufacturer of the mouse diet were changed. Over the same period, the IOP of A/J also increased dramatically, from 9.4 ± 0.5 mmHg (n = 11) in 1996 to 14.2 ± 0.4 mmHg (n = 19) in 1997. The increased IOP in A/J also was reproducible, with the average IOP of mice assessed in the year 2000 being 14.5 ± 0.4 mmHg (n = 16). Importantly, the IOPs of strains B6 and C3H/HeJ did not change during this time (B6, 12.3. ± 0.5 mmHg in 1996 and 12.4 ± 0.3 mmHg in 1997, n= 10 and 14; C3H/HeJ, 13.7 ± 0.8 mmHg in 1996 and 13.6 ± 0.2 mmHg in 1997, n = 9 and 19).
Effect of age on IOP
We focused on the commonly used B6, 129P3/J and C3H/HeJ strains to determine the effects of age on IOP (Figure ). In B6, age had a significant effect on IOP (P < 0.001). IOP was slightly decreased at both 12 months (12.2 ± 0.2 mmHg) and 19 months (12.2 ± 0.3 mmHg) compared to 3 months (13.1 ± 0.3 mmHg) and 7 months (13.3 ± 0.3 mmHg). Although the decrease was of a similar level to the variation observed in 3 month old mice (see Figure ), the IOPs of control young mice (see Methods) analyzed at the same times as the various B6 age groups did not decrease. For example, control mice analyzed at the same time as the 3 month, 12 month and 18 month B6 age groups had IOPs of 13.0 ± 0.2 mmHg (n = 14), 13.3 ± 0.2 mmHg (n = 14), 13.7 ± 0.4 mmHg (n = 12), respectively. IOP was even lower in the 24 month B6 mice (10.8 ± 0.4 mmHg), and again the average IOP of young controls measured at the same time was not changed (13.5 ± 0.2, n = 12).
Figure 4 IOP changes with increasing Age. Mean IOP ± SEM is shown for the indicated strain at different ages. IOP decreased significantly in strains C57BL/6J and 129P3/J but not C3H/HeJ. The C57BL/6J and 129P3/J groups consisted of approximately equal (more ...)
In strain 129P3/J, IOP did not differ significantly with age between 3 and 14 months but was lower in 18 month old mice (P < 0.001 compared to all younger ages, Figure ). Despite a 1 mmHg dip in IOP at 8 months, there were no significant IOP differences between C3H/HeJ mice at each age tested (P = 0.2 for age). Although the effect of age has not been thoroughly assessed in other strains, no obvious age-related differences have been identified in other strains analyzed at multiple ages except for the glaucomatous DBA/2J and AKXD-28/Ty strains [14
Effect of sex on IOP
Although we have not rigorously assessed the effect of sex on IOP in many strains, sex specific differences have not been detected in the majority of strains for which both sexes have been analyzed, and have proven inconsistent even within an individual strain analyzed multiple times. Strains B6 and 129P3/J have been extensively evaluated at multiple ages between 3 and 24 months of age. Sex differences were always absent in strain 129P3/J and typically absent in strain B6. In strain B6, however, males infrequently had significantly higher IOP than females. For example, in one experiment, B6 males had an average IOP of 14.2 ± 0.3 mmHg (n = 12) whereas the average IOP of females was 13.1 ± 0.3 mmHg (n = 12, P = 0.01). If real, this sporadic sex difference was not dependent on age, sometimes occurring in a group of B6 mice at a particular age and sometimes not occurring in a separate group of the same age.
Anesthesia protocol avoids IOP alteration and allows detection of diurnal differences
All IOPs were assessed using an anesthetic regime of 99 mg/kg ketamine and 9 mg/kg xylazine (defined as 1X). Initial experiments suggested that an almost identical dose (100 mg/kg ketamine and 9 mg/kg xylazine) of anesthesia had no effect on IOP during the experimental period with IOP being measured as soon as possible after the mouse was unconscious, typically minutes. [13
]. To further assess the effects of anesthesia, we measured IOP in groups of genetically identical B6 mice subjected to different doses (1X, 1.5X and 2X) at 5 and 25 minutes after administration (Figure ). For all doses, IOP decreased by 25 minutes (P = 0.005 for time). The greater the dose the greater the decrease in IOP. At the 5 minute measurement, however, IOP was the same using all doses suggesting that the anesthetic effect on IOP had not yet occurred. To identify any early window when it may be possible to assess IOP without an obvious anesthetic effect, 195 mice of strain B6 were anesthetized with the 1X dose and IOP was measured at 1 minute time points between 4 and 12 minutes after administration (Figure ). The mean IOP of groups analyzed at each time point did not differ (P = 0.9) indicating that the IOP depressing effect of anesthesia occurs later than 12 minutes after administration. Similar results were obtained using 161 strain 129P3/J and 145 strain DBA/2J mice with the 1X dose (129P3/J, P = 0.1; DBA/2J, P = 0.2). In support of a later effect of anesthesia (since general anesthesia is reported to mask diurnal variation in IOP [21
]), we identified increased IOP during the dark compared to the light period of the day in several tested strains (Figure ). In these experiments, IOP measurements were made between 5 and 12 minutes after administration of anesthesia.
Figure 5 No effect of 1X anesthetic dose within 12 minutes of administration. A Mean IOP ± SD is shown for C57BL/6J mice at 5 and 25 minutes after administration of various doses of anesthetic. The 1X dose consisted of 99 mg/kg ketamine and 9 mg/kg xylazine. (more ...)
Figure 6 IOP is increased during the dark compared to light periods in several strains. Mean IOP ± SEM is shown for the indicated strains at measurement during the light (open bars) or dark (filled bars) periods of the day. All mice were 2 to 4 months (more ...)
Blood pressure does not correlate with IOP
An initial study of the relationship between blood pressure and IOP in mice did not detect a good correlation (R2 = 0.1, Figure ).
Figure 7 No correlation between blood pressure and IOP. There is no strong correlation between the average systolic blood pressure of each strain and the average IOP of that strain (R2 = 0.1). All mice were female and 2 to 4 months old, with the exception that (more ...)
Myoc alleles do not associate with the magnitude of IOP
Mutations in the myocilin gene (MYOC
) cause human glaucoma. To determine if allelic variation in the mouse Myoc
gene associated with IOP in mouse strains, we analyzed the gene in an assortment of strains with different IOPs. Two alleles were identified. One of these alleles had a 12 nucleotide insertion in the promoter region (ccagagcagggt, between positions -340 and -341) compared to the previously published sequence and is called the insertion allele. The other allele was identical to the published sequence [22
]. The insertion allele also had a previously reported substitution (A to G, Thr164Ala) in exon 1 and several other single base changes in the promoter region [22
]. The presence or absence of this allele does not associate with IOP as it is present in strains with a range of IOPs (Figure ).
Figure 8 Myoc alleles do not associate with IOP. Sequencing identified two alleles of Myoc in these mouse strains (see text). Mouse strains homozygous for an allele having a 12 bp insertion in the promoter region are shown as open bars and strains homozygous (more ...)
Genetic alterations and IOP
The Y chromosome has been implicated in strain specific blood pressure differences in rats [23
]. To test if the Y chromosome of strain 129/Ola alters IOP in relation to that of strain B6, we compared the IOPs of pure B6 males and consomic B6 males that had the 129P2/Ola Y chromosome (backcrossed for 11 generations). No differences in IOP were detected between these groups of mice (Figure , P = 0.1).
Figure 9 Effects of genetic alterations. Mean IOP ± SEM is shown. There is no difference in IOP between 3 to 4 month old males with either the 129P2/Ola (n = 18) or C57BL/6J (n = 20) Y chromosome (Y Chr). The 129 Y Chr had been backcrossed to strain C57BL/6J (more ...)
To test if deficiency of carbonic anhydrase II leads to decreased IOP, we analyzed mice of a B6 background that were genetically similar but with normal or mutant alleles of the Car2
]. There was no difference in IOP between normal and mutant mice (Figure , P = 0.5).
To test if genetic perturbations that cause obesity and diabetes can alter IOP, we compared mice that were genetically similar but were either homozygous or heterozygous for a leptin receptor mutation (db
) that results in obesity and diabetes before 4 months of age on the C57BLKS/J strain background used [27
]. IOP was modestly but significantly elevated in obese, diabetic homozygous mutants (14.7 ± 0.3 mmHg) compared to non-obese, non-diabetic heterozygotes (13.4 ± 0.4 mmHg, P < 0.01, Figure ).
To determine if albinism alters IOP, we analyzed B6 mice that were either pigmented or albino. The albino mice were homozygous and coisogenic for a mutant allele of tyrosinase (Tyrc-2J) that arose on the otherwise pigmented B6 background. In 2 month old mice, homozygosity for Tyrc-2J resulted in increased IOP (14.2 ± 0.4 mmHg) compared to wild type, pigmented mice (12.4 ± 0.3 mmHg, P < 0.0001). The same was true for independent cohorts of mice of different ages that were analyzed at different times (Figure ). In contrast to pigmented B6 mice (Figure ), the IOPs of the albino B6 mice were not increased at measurement during the dark compared to light period of the day (P = 0.6, Figure ).
Figure 10 Tyr mutation results in increased IOP and altered diurnal changes A. Different cohorts of C57BL/6J mice that are homozygous for the spontaneous Tyrc-2J mutation have higher IOP than their normal counterparts at all tested ages. All groups consisted of (more ...)