Optic Nerve Damage in Mice with a Targeted Type I Collagen Mutation

Abstract

purpose. Transgenic (Col1a1 ^r/r) mice gradually develop elevated intraocular pressure (IOP) with open angles. The present study was undertaken to evaluate optic nerve axonal loss with time in these mice.

methods. The IOP of transgenic (Col1a1 ^r/r) mice and control wild-type (Col1a1 ^+/+) mice was measured at 7, 12, 16, 24, 36, and 54 weeks of age using a microneedle method. Transgenic Col1a1 ^r/r and control Col1a1 ^+/+ mice at 24 and 54 weeks of age were randomly selected and their optic nerves were processed conventionally for electron microscopy. Optic nerve cross-sections were collected 300 μm posterior to the globe. Low (200X) and high (10,000X) magnification images were collected systematically and were masked before analysis. For each nerve, cross-sectional area was measured in low magnification images, and axonal number was counted in high magnification images.

results. Mean IOP of the transgenic Col1a1 ^r/r mice was significantly higher than that of the control Col1a1 ^+/+ mice at 16, 24, 36, and 54 weeks by 21%, 42%, 41%, and 33% respectively (P < 0.05). The mean axonal density and total axonal number in the transgenic Col1a1 ^r/r mice at 54 weeks of age (n = 10) was significantly less than those in the control Col1a1 ^+/+ mice at 24 weeks (n = 5) and 54 weeks (n = 5; P = 0.0081 and P = 0.020, respectively, analysis of variance, P < 0.05 for pair-wise comparisons). The mean axonal density and total axonal number in the transgenic Col1a1 ^r/r mice at 54 weeks also were significantly less than in the transgenic Col1a1 ^r/r mice at 24 weeks (n = 10). Mean axonal loss between 24 and 54 weeks of age in the transgenic Col1a1 ^r/r mice was 28.7%.

conclusions. Transgenic Col1a1 ^r/r mice develop sustained elevation of IOP and progressive optic nerve axon loss. This suggests that these mice may be useful as a mouse model of primary open angle glaucoma as well as for assessing the relationship between collagen type I metabolism and optic nerve axon loss.

High intraocular pressure (IOP) is a leading risk factor for primary open angle glaucoma. ^{1 2} Previous investigations of the mechanism of glaucomatous optic neuropathy have studied animals, including monkey ^{3 4} and rat, ^{5 6 7 8 9 10 11} that have had elevated IOP induced experimentally. Although these models have provided important information to clarify the relationship of retinal ganglion cell and axon loss with elevated IOP, the molecular mechanism(s) underlying these losses remains poorly understood. 

Interest in the mouse as a model system to study glaucoma has emerged with the maturation of mouse transgenic technology that offers the possibility of investigating the effects of single or multiple gene alterations on glaucomatous axon loss in vivo. ¹² Three mouse strains with spontaneous IOP elevation and associated optic nerve axon loss have previously been described. ^{13 14 15} Within these strains, IOP elevation was variable and associated with the formation of anterior synechiae and closed angle obstruction of aqueous outflow. 

A transgenic mouse strain with a targeted mutation in the gene for the α1 subunit of collagen type I had a spontaneous and gradual IOP elevation. ¹⁶ This mutation is positioned at the consensus cleavage site for matrix metalloproteinase (MMP)–1 and inhibits hydrolysis of the collagen type I α1 subunit by endogenous MMPs. An age-dependent accumulation of collagen type I has been observed within several different collagen-containing tissues of these mice. ^{17 18 19} In this strain, mean IOP became significantly higher than in the wild-type mice by 18 weeks of age and progressed to a 44% IOP elevation by 24 weeks of age. ¹⁶ In addition, the anterior chamber angle remained open in these transgenic mice. ¹⁶ If there is gradual optic nerve damage in this mouse strain, it may provide an open angle glaucoma model. The present study was undertaken to determine whether these mice have optic nerve axon loss. 

Materials and Methods

Animals

Animals used in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Transgenic mice, designated B6;129S4-Col1a1 ^tm1Jae (Col1a1 ^r/r), have a targeted mutation of the gene for procollagen type I α1 subunit sequence: Gln₇₇₄ to Pro, Ile₇₇₆ to Met, Ala₇₇₇ to Pro, Val₇₈₂ to Ala, and Val₇₈₃ to Pro. ^{17 20} Breeding pairs of these mice and the corresponding control wild-type (Col1a1 ^+/+) were obtained from Jackson Laboratory (Bar Harbor, ME) and homozygous matings were conducted to expand the number of transgenic and wild-type mice. Homozygous transgenic Col1a1 ^r/r and control Col1a1 ^+/+ mice were used in this study. Mice were 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 and 12-hour dark cycle. All mice were fed ad libitum. Additional (Col1a1 ^r/r) mice of various ages were a gift from Simon John of The Jackson Laboratory, Bar Harbor, ME. 

IOP Measurements

The IOP of the transgenic Col1a1 ^r/r and control Col1a1 ^+/+ mice was measured at 7, 12, 16, 24, 36, and 54 weeks after birth with a microneedle method as previously described by Aihara et al. ^{16 21} Mice were anesthetized with an intraperitoneal injection of a solution containing ketamine (100 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (9 mg/kg, TranquiVed; Vedco, Inc., St. Joseph, MO). A fluid-filled glass microneedle connected to a pressure transducer was inserted through the cornea into the anterior chamber to measure IOP. 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 diurnal variation of IOP. 

Fixation and Processing of the Optic Nerve

At 24 and 54 weeks of age, the transgenic Col1a1 ^r/r mice and control Col1a1 ^+/+ mice were selected at random, and optic nerve tissue was processed conventionally for electron microscopy as previously described. ²² The 24-week-old mice were studied to assess whether total axon number at this time point was similar in the transgenic Col1a1 ^r/r mice and control Col1a1 ^+/+ mice. A previous study of this mouse strain found that this time point typically was approximately 6 weeks after maximal IOP elevation developed. ¹⁶ Because another previous study found optic nerve axon loss after experimental induction of elevated IOP was negligible at 6 weeks, but significant at 12 weeks, ²² assessment of axon survival in the present study 6 weeks after attainment of maximal IOP elevation was done to provide a baseline for the evaluation the effect of sustained IOP elevation on optic nerve axon survival. The 54-week-old mice were studied to assess the effect of sustained IOP elevation on axon loss. Briefly, the mice were anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg, Nembutal; Abbott Laboratories, North Chicago, IL) and exanguinated by perfusion with mammalian Ringer’s solution containing lidocaine hydrochloride (0.1 mg/mL, Xylocaine; Astra USA, Inc., Westborough, MA) and heparin sodium (500 unit/mL, Heparin; Elkins-Sinn, Inc., Cherry Hill, NJ). Transcardial perfusion was then continued with fixative (approximately 20 mL of 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.15 M cacodylate buffer). The optic nerves were dissected and placed in this fixative overnight. The optic nerves were postfixed in 1% osmium tetroxide, stained in 2% uranyl acetate, dehydrated in ethanol and acetone, and embedded in epoxy resin (Durcupan; Electron Microscopy Sciences [EMS], Fort Washington, PA). Ultrathin sections were cut perpendicularly to the long axis of the optic nerves on an ultramicrotome, and placed on Formvar-coated slot grids. These sections were obtained at approximately 300 μm posterior to the nerve emanation from the globe. Sections were counterstained with 1% uranyl acetate and Sato lead, and viewed with a JEOL 12,000 EX electron microscope. 

Optic Nerve Axon Counting

The number of axons in the mouse optic nerves was assessed according to the method developed by Williams et al. ²³ with minor modifications. ²² For each optic nerve cross-section, electron micrographs were taken at low magnification (×200) to measure the area of the optic nerve cross-section. Then a series of twenty micrographs were taken at high magnification (×10,000) in a square lattice pattern in the following positions within the optic nerve: center, four micrographs; mid-periphery, eight micrographs; peripheral margin, eight micrographs. No adjustments in position were made with respect to the tissues including blood vessels and glial cells. To confirm the true magnification, calibration grids (1000 mesh/inch, No. 79,525–01; EMS, Fort Washington, PA, and 2160 lines/mm, No. 206; Ted Pella, Redding, CA) were photographed at the same low and high magnifications. 

Electron micrographs were digitized using a Peltier-cooled high-resolution CCD camera (CH250; Photometrics, Inc., Tucson, AZ) and magnified at ×4 in the course of digitizing. The effective magnifications were therefore ×800 at low magnification, and ×40000 at high magnification. The identity of the digitized images was masked before analysis, and each image was analyzed using image-processing software (NIH Image, Version 1.62; public domain software). The area of the optic nerve cross-section was measured three times by outlining its outer border, and the mean of these measurements was used for subsequent calculations. To measure axonal density, a counting frame (7 × 8 cm) on the high magnification image was traced, and survival myelinated and unmyelinated axons within the frame and intersecting the upper and left edges were marked and counted manually using standard unbiased counting rules. ²⁴ The total area counted in the twenty micrographs analyzed for each nerve was 1120 μm². This corresponded to approximately 2% of the total nerve cross-sectional area. Axon profiles that did not contain neurofilaments were excluded from the counts because they possibly represented degenerating axons. The mean axonal density was calculated, and the total axonal number was estimated by multiplying the mean density by the area of the optic nerve cross-section. In a prior study, we found that the measurements of total axon number using this protocol within the three adjacent sections of the same optic nerve had coefficients of variation < 1.4%. ²² This indicated that this counting method could identify differences in the total axon number that exceeded 1.5%. 

Statistical Analysis

The difference between mean IOP was statistically analyzed by the two-way analysis of variance (ANOVA) and the Bonferroni/Dunn method for pair-wise comparisons. These comparisons included both animals in which IOP had been followed since birth and animals that had been received as adults. The one-way ANOVA and the Bonferroni/Dunn method for pair-wise comparisons were used for evaluation of optic nerve damage. P < 0.05 was considered to be statistically significant. 

Results

Intraocular Pressure

Mean IOP in the transgenic Col1a1 ^r/r mice and control Col1a1 ^+/+ mice from 7 to 54 weeks of age are shown in Figure 1 , and differences between these mice are shown in Table 1 . IOP of Col1a1 ^r/r mice was significantly higher than IOP of Col1a1 ^+/+ mice at 16, 24, 36, and 54 weeks by 21%, 42%, 41%, and 33%, respectively (P < 0.0001, two-way ANOVA, P < 0.0001 for pair-wise comparisons, Bonferroni/Dunn method). There was no significant gender difference in IOP. 

Optic Nerve Damage

The optic nerve axonal density and total axon number were not significantly different in 24-week-old transgenic Col1a1 ^r/r mice and control Col1a1 ^+/+ mice (Table 2 and Fig. 2 ). In contrast to the 24-week-old mice, the mean axonal density and in the transgenic Col1a1 ^r/r mice at 54 weeks of age (10 eyes) was significantly lower than that in the control wild-type Col1a1 ^+/+ mice at 24 weeks (5 eyes) and 54 weeks (5 eyes) of age and in transgenic Col1a1 ^r/r mice at 24 weeks of age (10 eyes) (P = 0.0081, one-way ANOVA, P < 0.05 for pair-wise comparisons, Bonferroni/Dunn method, Fig. 2A ). The total axonal number in transgenic Col1a1 ^r/r mice at 54 weeks of age was also significantly less than that in the other three groups (P = 0.020, one-way ANOVA, P < 0.05 for pair-wise comparisons, Bonferroni/Dunn method, Fig. 2B ). There was no significant gender difference in axon loss. On average, there were 28.7% axonal loss and 33.4% axonal density decrease between 24 and 54 weeks of age in the transgenic Col1a1 ^r/r mice. 

Examination of the electron micrographs of the optic nerves from the 54-week-old Col1a1 ^r/r mice showed that the prevalence of axons with inclusion bodies and glial cell profiles within the optic nerve increased with the magnitude of axon loss (Fig. 3) . 

Discussion

The present results demonstrate that optic nerve axon loss occurs in transgenic Col1a1 ^r/r mice with elevated IOP. Specifically, axon loss at 54 weeks of age was less than that in the control wild-type Col1a1 ^+/+ mice. Although there was no significant difference of the axonal number between the control wild-type Col1a1 ^+/+ mice at 24 and 54 weeks of age, the transgenic Col1a1 ^r/r mice had approximately 30% axonal loss between 24 and 54 weeks of age. Moreover, the number of axons observed in the wild-type mice at 24 weeks and at 54 weeks of age as well as in the Col1a1 ^r/r mice at 24 weeks of age differed insignificantly from the number of axons we observed previously in untreated 18—20-week-old Black Swiss mice (59,597 ± 3112, n = 10). ²² In view of the open angle in the Col1a1 ^+/+ anterior chamber, ¹⁶ these observations suggest that Col1a1 ^+/+ mouse may be a useful open angle model of glaucomatous optic neuropathy. 

The accumulation of collagen type I throughout the transgenic Col1a1 ^r/r mouse eye may have both indirect and direct influences on the development of optic nerve damage. The normal mouse eye has a well-developed Schlemm’s canal ²⁵ and uveoscleral outflow pathway similar to that in human eye. ²⁶ Histologic analysis of Col1a1 ^r/r mice showed that the sclera is thicker and more immunoreactive for collagen type I than sclera from the eyes of control Col1a1 ^+/+ mice. ¹⁶ However, the angle remained open and no reorganization of trabecular outflow pathway structures was observed. These observations suggest that, though the basic pathway of aqueous outflow was unaltered, the facility of aqueous outflow may have become impaired by collagen accumulation within the trabecular and uveoscleral pathways. Further studies of aqueous dynamics within these mice, as recently reported in other mouse strains, ^{27 28} may further clarify the impact of the collagen type I mutation on aqueous outflow. 

Increased scleral collagen may have directly contributed to increased susceptibility of the optic nerve to damage by elevated IOP. An immunohistochemical study has shown that mouse optic nerve axons pass through a scleral canal that is surrounded by a ring of type I and type III collagen fibers. ²⁹ In view of the increased scleral collagen in Col1a1 ^r/r mice, it possible that collagen I accumulation within this ring may have stiffened the edge of the scleral canal in a manner similar to the increased skin rigidity that has been previously reported in the Col1a1 ^r/r mice. ²⁰ Whether this change occurs, whether there is a loss of tissue compliance at the optic nerve head, and whether there is associated increase in optic nerve damage susceptibility, should be studied further. 

The gradual development and maintenance of IOP elevation in Col1a1 ^r/r mice suggest it may be well-suited for investigations of gradual optic nerve damage. Additional study of the optic nerve in Col1a1 ^r/r mice at time points shorter and longer than 54 weeks may further clarify the relationship between axon loss and duration of IOP elevation. Nevertheless, IOP elevation in this model appeared to be more gradual than in other mouse models of glaucoma. For example, laser treatment to the limbus of NIH black Swiss mice induced a 91% increase of mean IOP that returned to baseline by 6 weeks after laser treatment and was associated with a loss of about two-thirds of total axons by 12 weeks after treatment. ^{22 30} Elevation of IOP in DBA/2J mice often occurs quickly with initiation between 8 and 12 months of age and maximal IOP greater than 30 mm Hg. ¹⁴ Similarly, elevation of IOP in AKXD28 mice typically occurs quickly with onset between 15 and 18 months of age and maximal IOP greater than 30 mm Hg. ¹⁴ In DBA/2J and AKXD28 mice, the optic nerve damage at the same age varies individually, ranging from mild to severe, and is observed in some but not all mice. ¹⁴ Danias and coworkers found IOP elevation in DBA/2NNia mice often occurs between 6 and 9 months of age and begins to decline at 12 months of age, but significant loss of retinal ganglion cells is not observed until 15 months of age. ¹⁵ However, the present study has shown IOP elevation in transgenic Col1a1 ^r/r mice begins by 5 months, plateaus at approximately 24 mm Hg, and remains moderately elevated at 1 year old (54 weeks of age). Moreover, the variability of axonal loss in transgenic Col1a1 ^r/r mice at 54 weeks of age may be lower than that in other mouse strains with IOP elevation. ^{14 15 22} In addition, these mice have no gross reorganization of anterior segment structure and the anterior chamber angle is open, although accumulation of collagen I has been observed in the sclera. ¹⁶  

In conclusion, the present study demonstrates that optic nerve axon loss in transgenic Col1a1 ^r/r mice, which develop sustained elevated IOP as they mature, is associated with age. This transgenic mouse may be a useful model for primary open angle glaucoma. 

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

 Submitted for publication September 11, 2003; revised January 17, 2004; accepted February 29, 2004.

 Disclosure: F. Mabuchi, None; J.D. Lindsey, None; M. Aihara, None; M.R. Mackey, 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.

 

Age-related changes of IOP in the 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 (P < 0.0001, two-way analysis of variance, P < 0.0001 for pair-wise comparisons, Bonferroni/Dunn method). Data points of these mice up to 36 weeks of age have been reported previously. ¹⁶ The data points at 54 weeks of age are new. The number at each data points means the number of eyes.

Table 1.

 

IOP in Transgenic Col1a1 ^r/r Mice and Control Wild-type Col1a1 ^+/+ Mice of Different Ages

	Age
		Week 7	Week 12	Week 16	Week 24	Week 36	Week 54
Transgenic Col1a1 r/r mice (eyes)	20	34	43	59	76	73
IOP of Col1a1 r/r [IOPt] (mm Hg; mean ± SD)	16.7 ± 0.8	20.7 ± 4.0	23.0 ± 2.5	22.7 ± 2.4	22.8 ± 3.4	21.5 ± 2.4
Control wild-type Col1a1 +/+ mice (eyes)	7	18	12	14	10	8
IOP of Col1a1 +/+ [IOPc] (mm Hg: mean ± SD)	15.2 ± 1.4	18.8 ± 1.9	19.0 ± 1.8	16.0 ± 0.9	16.1 ± 1.1	16.1 ± 1.6
Δ IOP (mm Hg) [IOPt− IOPc]	1.5	1.9	4.0	6.7	6.7	5.7
Δ IOP (%) [100 × (IOPt− IOPc)/IOPc]	10	10	21	42	41	33

Table 2.

 

Optic Nerves in Transgenic Col1a1 ^r/r Mice and Control Wild-type Col1a1 ^+/+ Mice of Different Ages

	Control Wild-type Col1a1 +/+ Mice (Age)			Transgenic Col1a1 r/r Mice (Age)
		Week 24 (5 eyes)	Week 54 (5 eyes)	Week 24 (10 eyes)	Week 54 (10 eyes)
Optic Nerve Cross-Sectional Area (μm2)	62183 ± 9710	65294 ± 10471	66166 ± 9693	70080 ± 17448
Mean Axon Density (number/μm2)	1.0 ± 0.21	0.96 ± 0.16	1.0 ± 0.23	0.66 ± 0.24
Total Axon Number (number/optic nerve)	60995 ± 9160	61537 ± 4677	65048 ± 12667	46621 ± 16067

Figure 2.

 

Age-related changes of mean axonal density (A) and total axonal number (B) of the optic nerve in the control wild-type Col1a1 ^+/+ mice (W24: 24 weeks of age, 5 eyes; W54: 54 weeks of age, 5 eyes) and transgenic Col1a1 ^r/r mice (T24: 24 weeks of age, 10 eyes; T54: 54 weeks of age, 10 eyes). The mean axonal density and in the transgenic Col1a1 ^r/r mice at 54 weeks of age was significantly lower than that in the control wild-type Col1a1 ^+/+ mice at 24 weeks and 54 weeks and in transgenic Col1a1 ^r/r mice at 24 weeks (P = 0.0081, one-way analysis of variance). The total axonal number in transgenic Col1a1 ^r/r mice at 54 weeks of age was also significantly less than that in the other three groups (P = 0.020, one-way analysis of variance). Error bar indicates SD *P < 0.05, Bonferroni/Dunn method.

Figure 3.

 

High magnification (original magnification, ×10,000) images of the optic nerve cross-section in the control wild-type Col1a1 ^+/+ mice at 24 weeks of age (A) and at 54 weeks of age (B), and transgenic Col1a1 ^r/r mice at 24 weeks of age (C), and 54 weeks of age with 22% (D), 33% (E), and 45% (F) of axon density decrease compared to the mean axon density in the control wild-type Col1a1 ^+/+ mice at 54 weeks of age. Final magnification, ×6400.

The authors thank Simon John, Jackson Laboratories, Bar Harbor, ME for helpful discussions regarding the care and breeding of the Col1a1 ^r/r mice.