Ocular Hypertension in Mice with a Targeted Type I Collagen Mutation

Abstract

purpose. To evaluate intraocular pressure (IOP) in transgenic mice with a targeted mutation in the gene for the α1 subunit of collagen type I.

methods. Homozygous B6; 129-Cola1 ^tm1Jae mice and corresponding wild-type mice were anesthetized. A fluid-filled glass microneedle connected to a pressure transducer was then inserted through the cornea into the anterior chamber to measure IOP. All measurements were made between 11:30 AM and 1:30 PM. The IOP of seven Col1a1 ^r/r and eight corresponding wild-type Col1a1 ^+/+ male mice was measured at 12, 18, 24, and 36 weeks after birth. The IOP of 5 to 24 additional Col1a1 ^r/r mice was measured at 7, 12, 18, 24, and 36 weeks after birth. The structure of the anterior segment and the distribution of collagen I were assessed by immunohistochemistry.

results. Mean IOP measurements of the control Col1a1 ^+/+ mice (IOP_c) at 12 and 18 weeks after birth were relatively constant at 18.9 ± 2.0 and 19.2 ± 1.9 mm Hg, respectively. Mean IOP then decreased to 15.8 ± 0.8 and 16.2 ± 1.2 mm Hg at 24 and 36 weeks, respectively. In contrast, mean IOP measurements in the transgenic (Col1a1 ^r/r) mice was 2.7 ± 3.4 mm Hg higher at 12 weeks and increased to a maximum of 23.6 ± 2.4 mm Hg at 24 weeks. The difference between mean IOP in these two groups gradually increased to a maximum of 4.8 mm Hg (30%) at 36 weeks and was significantly different from the control mice at both 24 and 36 weeks of age. No anterior segment abnormality was observed in Col1a1 ^r/r mice and no difference between the anterior segment appearance of Col1a1 ^r/r and Col1a1 ^+/+ mice was observed throughout the 36-week analysis period. However, collagen I immunoreactivity in sclera and associated structures was greater in Col1a1 ^r/r mice than in Col1a1 ^+/+ mice. When the mean IOP measurements from the additional Col1a1 ^r/r mice were included with these measurements, mean IOP at each age was 16.7 ± 0.8, 21.8 ± 3.9, 23.2 ± 2.8, 23.5 ± 2.4, and 22.1 ± 3.6 mm Hg, respectively. Mean IOP in the Col1a1 ^r/r mice was significantly higher than in the Col1a1 ^+/+ mice at 18, 24, and 36 weeks by 21%, 44%, and 36%, respectively (P < 0.05).

conclusions. These results demonstrate ocular hypertension in mice with a targeted type I collagen mutation and suggest there is an association between IOP regulation and fibrillar collagen turnover.

Fibrillar collagen, consisting of heterotrimers of the collagen type I and type III subunits, is a major component of structures within the trabecular meshwork and uveoscleral aqueous humor outflow pathways, including trabecular meshwork beams and the extracellular matrix of the ciliary muscle, the choroid, and sclera. ^{1 2 3 4} Aqueous humor contains several different matrix metalloproteinases (MMPs), members of a family of neutral proteases that on activation can cleave specific sites within extracellular matrix collagens and thereby initiate their turnover. ^{5 6} Potential interaction between these collagens and MMPs is suggested by the presence of immunoreactivity for MMPs within the trabecular meshwork, ciliary muscle, and sclera of normal human and monkey eyes. ^{1 5 6 7 8 9} In addition, there is substantial evidence that cells within the trabecular meshwork, ciliary muscle, and sclera, can synthesize MMPs. ^{7 8 9 10}  

The functional significance of MMPs within the outflow pathway tissues is suggested by experiments showing that introduction of activated MMPs into perfused human anterior segment organ cultures increases outflow facility. ¹¹ Exposure of these cultures to growth factors that stimulate MMP biosynthesis by trabecular meshwork cells also increases outflow facility. In contrast, introducing inhibitors of MMP activity into these perfusion cultures reduces outflow facility. ¹¹ Similarly, exposure of isolated human scleral organ cultures to drugs that stimulate endogenous production of MMPs also increases transscleral permeability to macromolecules. ^{10 12} These observations raise the possibility that controlled extracellular matrix turnover may be essential to the normal regulation of intraocular pressure (IOP). However, direct evidence of MMP-mediated collagen turnover in normal untreated eyes has not been found. 

To investigate the role of MMPs in IOP regulation in untreated eyes, the present study examined the time course of IOP changes in transgenic mice with targeted mutations in the gene for α1 subunit of collagen type I. This mutation codes for five amino acid substitutions adjacent to the normal MMP-1 cleavage site, and these substitutions completely block MMP-1 hydrolysis. ¹³ Recent experiments have confirmed that this cleavage site also is targeted by MMP-2. ^{14 15} Mice homozygous for this mutation develop normally into young adulthood. ¹⁶ With increasing age, however, these mice show development of marked fibrosis of the dermis, similar to human scleroderma. Also noted is impairment of postpartum involution of the uterus with persistence of collagenous nodules in the uterine wall. Collagen turnover in other tissues involves both MMP-mediated initiation of degradation and new collagen biosynthesis. ^{17 18 19} Thus, if turnover of fibrillar collagen within outflow tissues is associated with normal IOP regulation, then the impairment of MMP-mediated hydrolysis should result in a gradual accumulation of collagen type I within the outflow pathway tissues that could inhibit aqueous outflow and increase IOP. 

Methods

Mice

All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Transgenic mice, designated B6;129S4-Col1a1 ^tm1Jae (Col1a1 ^r/r), had a targeted mutation of the gene for procollagen type I α1 subunit (Col1a1) that yielded the following alterations in the amino acid sequence: Gln₇₇₄ to Pro, Ile₇₇₆ to Met, Ala₇₇₇ to Pro, Val₇₈₂ to Ala, and Val₇₈₃ to Pro. ^{13 16} Breeding pairs of these mice and the corresponding control wild-type (Col1a1 ^+/+) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Homozygous matings were conducted to expand the number of transgenic and wild-type mice. For the IOP analysis, we used homozygous male transgenic Col1a1 ^r/r and control (Col1a1 ^+/+) mice born of homozygous matings. Mice were bred and housed in clear cages covered loosely with air filters and containing white pine shavings for bedding. The environment was kept at 21°C with a 12-hour light-dark cycle. All mice were fed ad libitum. Additional Col1a1 ^r/r mice of various ages were a gift from Simon John (The Jackson Laboratory, Bar Harbor, ME). 

Anesthesia

After measurement of body weight, 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), prepared at room temperature. Animals were gently restrained in a plastic truncated cone (Decapicone; Braintree Scientific, Inc., Braintree, MA) to avoid stress, and anesthesia was administered with a 30-gauge needle. Each mouse was monitored carefully to assess the state of anesthesia. If the mouse displayed no response to pinching of the back skin, it was placed on the platform for measurement of IOP. 

IOP Measurement

After anesthesia, the anterior segments were examined with biomicroscopy, and then one eye was selected at random for measurement of IOP with a microneedle method described previously. ²⁰ IOP was measured during 4 to 8 minutes after anesthesia to minimize the influence of anesthesia on the pressure. The measurements were made between 11:30 AM and 1:30 PM, to minimize the influence of the 24-hour rhythm of IOP. The IOP data were masked during the measurement. The IOP of seven Col1a1 ^r/r and eight Col1a1 ^+/+ male mice born of the homozygous matings was measured at 12, 18, 24, and 36 weeks after birth. 

To address the possibility that elevation of IOP occurs adventitiously in a small colony, IOP was measured in different Col1a1 ^r/r and Col1a1 ^+/+ mice outside this initial group from 7 weeks of age. Hence, the total number of measured Col1a1 ^r/r mice was 12 at 7 weeks of age, 17 at 12 weeks, 22 at 18 weeks, 27 at 24 weeks, and 31 at 36 weeks. The total number of measured Col1a1 ^+/+ mice was seven at 7 weeks of age, eight at 12 weeks, eight at 18 weeks, 8 at 24 weeks, and eight at 36 weeks. 

Evaluation of Increase in IOP

IOP of Col1a1 ^r/r mice (IOP_r) and IOP of control (Col1a1 ^+/+) mice (IOP_c) were compared by t-test, and the percentage increase was calculated by the following formula: ΔIOP(%) = 100 × (IOP_r − IOP_c)/IOP_c. The difference between mean IOP at each age was statistically analyzed by the two-tailed Student’s t-test. The body weight of Col1a1 ^r/r and Col1a1 ^+/+ mice at each age was also compared by the two-tailed Student’s t-test. All data are presented as the mean ± SD. 

Histologic Analysis of Anterior Segment

Eyes of Col1a1 ^r/r and Col1a1 ^+/+ mice at 40 weeks of age were enucleated and fixed in 2% paraformaldehyde and 8-μm-thick frozen sections were mounted on glass slides. These sections were exposed to an antigen-retrieval solution (AR-10; BioGenex, San Ramon, CA) for 30 seconds at 70°C. The following procedures were accurately performed by a robotic staining system (Optimax; BioGenex). The sections first were blocked with casein for 30 minutes (PowerBlock, BioGenex) and then incubated for 3 hours at room temperature with biotinylated goat anti-human collagen I polyclonal antibody (Southern Biotechnology, Birmingham, AL; 1:100; two changes 1.5 hours each). After they were rinsed, the sections were exposed for 30 minutes to horseradish peroxidase-conjugated streptavidin (BioGenex). Each section was rinsed and incubated with the chromogen 3,3′-diaminobenzidine for precisely 10 minutes (HRP-DAB Super Sensitive Immunodetection System; BioGenex). As a negative control, sections were stained at the same time by the same procedure, except that the primary antibody was omitted. 

Results

IOP Changes in the Same Transgenic and Control Mice Observed for 36 Weeks

The changes in IOP and body weight among the initial group of Col1a1 ^r/r mice and control (Col1a1 ^+/+) mice are shown in Table 1 . Mean IOP measurements of the control Col1a1 ^+/+ mice were relatively constant at 18.9 ± 2.0 and 19.2 ± 1.9 mm Hg at 12 and 18 weeks of age, respectively. Mean IOP in these mice then decreased to 15.8 ± 0.8 and 16.2 ± 1.2 mm Hg at 24 and 36 weeks, respectively. In contrast, mean IOP in the transgenic (Col1a1 ^r/r) mice started higher by 2.7 ± 3.4 mm Hg than the control mice at 12 weeks. There was no significant difference in IOP between transgenic and control mice at this time point. Later, the IOP of transgenic mice increased to a maximum of 23.6 ± 2.4 mm Hg at 24 weeks. The difference in IOP in these two groups gradually increased to a maximum of 4.8 mm Hg (30%) at 36 weeks. IOP in the transgenic mice was significantly higher than in the control mice at both 24 and 36 weeks of age (P < 0.05). No anterior segment abnormality was observed among the Col1a1 ^r/r mice by biomicroscopy. The angle was open, and there was no difference between the depth of the anterior chamber in Col1a1 ^r/r and Col1a1 ^+/+ mice. The mean body weight of Col1a1 ^r/r and Col1a1 ^+/+ mice was similar throughout the 36-week duration of the investigation, except for a 9.8% difference recorded at 18 weeks. 

IOP Differences among Transgenic and Control Mice of Different Ages

Mean IOP and body weight among the total Col1a1 ^r/r and Col1a1 ^+/+ mice from 7 to 36 weeks of age are shown in Figure 1 and Table 2 . After including the IOP measurements from outside Col1a1 ^r/r mice with the bred Col1a1 ^r/r, the mean of these measurements (IOP^′ _r) was 16.7 ± 0.8 mm Hg at 7 weeks of age, 21.8 ± 3.9 mm Hg at 12 weeks, 23.2 ± 2.8 mm Hg at 18 weeks, 23.5 ± 2.4 mm Hg at 24 weeks, and 22.1 ± 3.6 mm Hg at 36 weeks. After including the IOP measurements from the outside Col1a1 ^+/+ mice with the bred Col1a1 ^+/+ mice, the mean of these measurements (IOP^′ _c) was 15.2 ± 1.4 mm Hg at 7 weeks of age, 18.9 ± 2.0 mm Hg at 12 weeks, 19.2 ± 1.9 mm Hg at 18 weeks, 15.8 ± 0.8 mm Hg at 24 weeks, and 16.2 ± 1.2 mm Hg at 36 weeks. There was no statistically significant difference between IOP^′ _r and IOP^′ _c at 7 and 12 weeks of age. However, IOP^′ _r was significantly higher than IOP^′ _c at 18, 24, and 36 weeks by 21%, 44%, and 36%, respectively (P < 0.05). The average of the differences between percentage increase in IOP relative to control mice in the bred Col1a1 ^r/r and the total Col1a1 ^r/r mice for weeks 12 (+1%), 18 (+3%), 24 (−4%), and 36 (+6%) was 1.5% (data from Tables 1 and 2 ). Hence, the addition of the IOP measurements from the outside transgenic mice minimally changed the results obtained with the bred mice alone. Thus, it is unlikely that the differences in IOP between the transgenic and control mice reflected a chance selection of an unusual cohort, but rather reflected the difference in genetic background. In contrast, the mean body weight of the total bred and outside Col1a1 ^r/r mice together was less than that of bred Col1a1 ^+/+ mice alone at 18, 24, and 36 weeks. 

Histologic Analysis of Anterior Segment and Collagen I Distribution

There was no structural difference between anterior segments of Col1a1 ^r/r and Col1a1 ^+/+ mice (Fig. 2) . However, collagen I immunoreactivity in conjunctiva, subconjunctival tissue, and sclera was greater in Col1a1 ^r/r mice than in Col1a1 ^+/+ mice. 

Discussion

The present results indicate that the difference between IOP in Col1a1 ^r/r mice and the corresponding wild-type mice gradually increased with age. Because the effect of this mutation is probably restricted to inhibition of MMP-mediated hydrolysis of collagen type I, this observation supports our hypothesis that the turnover of fibrillar collagen in normal untreated eyes is involved in the regulation of IOP. Moreover, the accumulation of collagen I in the anterior segment of Col1a1 ^r/r mice was observed by immunohistochemical analysis. Although the physiological effect of the accumulation of collagen I on outflow pathway is still unknown, these data are consistent with the possibility that the ocular hypertension reflects increased resistance in the outflow pathway. 

Two potential sources of variation were addressed within the experimental design. First, variation due to different amounts of stress as the animals matured or variations in genetic background among the cohorts measured at each time point were minimized in the first data set by observing IOP in the same set of mice as they matured. The similarity of these mice at each time point is suggested by the minimal variation in body weight. It is possible, however, that the group of homozygous transgenic animals selected for the first data set represented a subset with abnormally elevated IOP (i.e., it may not have been representative of typical Col1a1 ^r/r mice). Thus, additional measurements of Col1a1 ^r/r mice from outside the original group were combined with the measurements of the first set for each of the age groups. These mice represented from 59% to 79% of the total transgenic mice analyzed at each time point and, in many cases, the individuals contributed to measurements of just two or three of the age groups. Thus, if the mice of the first group were not representative, the addition of the measurements in outside mice may have significantly altered the results. However, the ΔIOP(%) differences observed when the outside mice data were included were similar to the results obtained when the data from the outside mice had been excluded, which indicates that the increasing difference in IOP between the transgenic and control mice was related to age. In contrast, differences in mean body weight at 18, 24, and 36 weeks of age were significantly altered by the addition of the outside mice data. Hence, the relationship of this parameter to increasing IOP in Col1a1 ^r/r mice appears to be less important than age, because the IOP measurements were not significantly influenced by the body weight variations. These observations collectively indicate that the differences in IOP in the transgenic mice reflect a specific effect of the Col1a1 gene product rather than a nonspecific effect due to chance selection of the subjects used within the original analysis. 

One consideration important to the interpretation of the results is that differences may exist between the expression of MMPs within the various tissues of mice and humans. For example, studies in humans conducted to date have not identified MMP-1 within adult rodent tissues, although an MMP-1 orthologue appears to be present in certain tissues during embryonic development. ²¹ This has led to the suggestion that MMP-13 may serve many of the same functions as MMP-1 in humans and other primates. ^{21 22} This may also explain why the targeted mutation within the helical region of the α1 subunit of collagen type I in the mice used in the present study was not lethal, as fibrillar collagen remodeling is essential for the later phases of embryonic morphogenesis. Specifically, MMP-13, unlike MMP-1, can cleave another site within the collagen molecule that may be sufficient to mediate the collagen remodeling that is essential for development. ¹⁶ In addition, it has recently been demonstrated that MMP-2 can cleave the same site within the collagen type I helical region that is targeted by MMP-1, though less efficiently than MMP-1. ^{14 15} Thus, cleavage of collagen type I in may in certain circumstances be mediated by MMP-2, and this may be more important in rodents than in humans. 

Further investigations of MMPs within rodents will clarify both the identity of MMPs involved within remodeling events within this genus. Nevertheless, the present results indicate that cleavage of the helical region of the α1 subunit of collagen type I is an essential ongoing process relevant to the regulation of IOP in normal untreated mouse eyes and, although the involvement of specific MMPs may vary across species, this process may be essential in regulation of IOP in human eyes, as well. 

It has been shown that skin-thickening in the Col1a1 ^r/r mice progresses with age. ¹⁶ This is consistent with the view that the transgenic alteration inhibits collagen hydrolysis associated with normal collagen turnover but does not alter ongoing biosynthesis of new collagen. In the present study, mean IOP in these mice was 21.6 mm Hg at 12 weeks of age and increased to 23.6 mm Hg by 24 weeks. Further supporting that elevation of IOP is progressive in these mice is the 31% increase in mean IOP of Col1a1 ^r/r mice between 7 and 12 weeks of age (P < 0.03, Student’s t-test). Thus, the increasing IOP in Col1a1 ^r/r mice with age is consistent with the accumulation of collagen within the routes for aqueous humor outflow. 

Immunohistochemical evidence obtained in the present study supports a link between collagen type I accumulation in the sclera of the Col1a1 ^r/r mice and increased IOP. Because the sclera is a distal component of the uveoscleral outflow pathway in the mouse, ²³ increased collagen type I within the sclera may impede uveoscleral outflow. Other outflow structures also may contain increased collagen type I; however, we could not assess this in frozen sections, either because these structures were too small or because they contained substantial pigment. Hence, immunoelectronmicroscopy is necessary to determine whether there are also alterations of collagen type I in other outflow pathway structures. 

The present study found an age-dependent decrease in IOP in the control Col1a1 ^+/+ mice. Age-dependent reductions of IOP have been reported in other mouse strains, including C57/B6, 129P3/J, and C3H/HeJ. ²⁴ Thus, an age-related reduction in IOP may be a natural feature of the control Col1a1 ^+/+ mice. In contrast, IOP in the transgenic Col1a1 ^r/r mice initially increased and then was relatively stable over the next 6 months. This stability may reflect the net effect of two processes. Thus, the normal events mediating the age-dependent reduction in IOP in the control Col1a1 ^+/+ mice may be balanced in the transgenic Col1a1 ^r/r mice by the effect of increased collagen type I on aqueous outflow. 

An important limitation of these results is they do not determine whether the effect of the Col1a1 mutation on IOP reflects alterations of trabecular meshwork outflow, uveoscleral outflow, or of both outflow pathways. Several studies indicate that collagen remodeling by MMPs can alter facility through each of these pathways. ^{10 11 12} Mice have a well-developed Schlemm’s canal. ²⁵ Moreover, uveoscleral outflow has recently been demonstrated within mouse eyes by experiments showing that intracamerally injected fluorescent dextran appears sequentially within the ciliary muscle, choroid, and equatorial sclera. ²³ Hence, studies are presently in progress to assess both conventional and uveoscleral outflow and to determine sites of collagen accumulation within the eyes of the mice used in the present study. 

In conclusion, the results of the present study demonstrate the presence of ocular hypertension in mice with a targeted type I collagen mutation and suggest that there is an association between regulation of IOP and turnover of fibrillar collagen. 

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

 Submitted for publication July 27, 2002; revised September 20 and October 17, 2002; accepted October 28, 2002.

 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.

 

Table 1.

 

IOP in the same Col1a1 ^r/r and Control Mice Over Time

	Age
		Week 12	Week 18	Week 24	Week 36
IOP of Col1a1 r/r [IOPr] (mm Hg; n = 7)	21.6 ± 3.4	22.6 ± 3.6	23.6 ± 2.4	21.0 ± 2.2
IOP of Col1a1 +/+ [IOPc] (mm Hg; n = 8)	18.9 ± 2.0	19.2 ± 1.9	15.8 ± 0.8	16.2 ± 1.2
IOP (mm Hg) (IOPr− IOPc)	2.7	3.4	7.6*	4.8*
IOP (%) [100× (IOPr− IOPc)/IOPc]	14	18	48*	30*
Body weight of Col1a1 r/r (g)	29.3 ± 1.3	31.4 ± 1.8*	35.8 ± 2.6	40.8 ± 3.4
Body weight of Col1a1 +/+ (g)	30.3 ± 2.0	34.8 ± 2.3	35.7 ± 2.1	41.3 ± 5.1

Figure 1.

 

Age-related changes of IOP in total transgenic Col1a1 ^r/r mice (•) and control Col1a1 ^+/+ mice (○). All IOP measurements are shown as the mean ± SD. *Significant difference between measurements of Col1a1 ^r/r and Col1a1 ^+/+ mice (t-test: P < 0.05).

Table 2.

 

IOP in Col1a1 ^r/r Mice and Control Mice of Different Ages

	Age
		Week 7	Week 12	Week 18	Week 24	Week 36
Col1a1 r/r mice (n)	12	17	22	27	31
IOP of Col1a1 r/r [IOP′ r] (mm Hg)	16.7 ± 0.8	21.8 ± 3.9	23.2 ± 2.8	23.5 ± 2.4	22.1 ± 3.6
Col1a1 +/+ mice (n)	7	8	8	8	8
IOP of Col1a1 +/+ [IOP′ c] (mm Hg)	15.2 ± 1.4	18.9 ± 2.0	19.2 ± 1.9	15.8 ± 0.8	16.2 ± 1.2
ΔIOP (mm Hg) [IOP′ r− IOP′ c]	1.5	2.9	4.0*	7.7*	5.9*
ΔIOP (%) [100× IOP′ r− IOP′ c/IOP′ c]	10	15	21*	44*	36*
Body weight of Col1a1 r/r (g)	23.6 ± 1.7*	27.6 ± 2.3	29.8 ± 2.0*	32.6 ± 2.5*	36.0 ± 5.2*
Body weight of Col1a1 +/+ (g)	19.1 ± 2.4	30.3 ± 2.0	34.8 ± 2.3	35.7 ± 2.1	41.3 ± 5.1

Figure 2.

 

Expression of collagen type I in the anterior segment of control Col1a1 ^+/+ (A) and transgenic Col1a1 ^r/r (B) mouse eyes. (C) Negative control staining. Note the increased collagen type I immunoreactivity in the sclera and conjunctiva. cor, cornea; con, conjunctiva; scl, sclera; ret, retina; cil, ciliary body. Original magnification, ×100.

The authors thank Simon John, PhD (The Jackson Laboratories, Bar Harbor, ME) for helpful discussions regarding the care and breeding of the Cola1 ^r/r mice.