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Optic Nerve Damage in Experimental Mouse Ocular Hypertension

¹From the Hamilton Glaucoma Center, the ²National Center for Microscopy and Imaging Research, and the ³Department of Neurosciences, University of California San Diego, La Jolla, California.

	Abstract

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PURPOSE. To evaluate optic nerve damage in mice after laser-induced ocular hypertension.

METHODS. Ocular hypertension was induced unilaterally in 13 NIH Black Swiss mice by laser photocoagulation of the limbus. Over the following 12 weeks, intraocular pressure (IOP) was measured at regular intervals by the microneedle method. The optic nerves of these mice and of seven normal untreated mice were then processed conventionally for electron microscopy, and cross sections of the nerve 300 µm posterior to the globe were collected. Low- and high-magnification images were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs, and axon and glia numbers were counted in high-magnification micrographs.

RESULTS. In normal untreated mice, the average number of axons was 59,597 ± 3,112 (mean ± SD). Variation among these measurements was 5.7% ± 3.9%. After laser treatment, the duration of high IOP ranged from 2 to 12 weeks (6.2 ± 3.6 weeks, mean ± SD). The mean IOP in the treated eyes was 1.3 times greater than the mean IOP in the control eyes (P = 0.0012). The maximum IOP in the treated eyes was 1.6 times greater than that observed in the control eyes (P < 0.0001). The optic nerve cross-sectional area, mean axon density, and total number of axons in the treated eyes were significantly less than in the control eyes (28.5% ± 23.4%, 57.8% ± 37.8%, and 63.1% ± 38.1%, respectively; P < 0.005 for each). The decrease in optic nerve cross-sectional area and the positive integral of elevated IOP and duration of IOP elevation correlated significantly with total axon loss (r² = 0.79, P < 0.0001 and r² = 0.36, P = 0.040, respectively). The number of astrocytes per cross section of optic nerve was significantly greater in the treated eyes than in the control eyes (P = 0.014).

CONCLUSIONS. Laser-induced ocular hypertension in mouse eyes can induce optic nerve axon loss that correlates with the magnitude and duration of elevated IOP.

A procedure to induce elevated IOP in normal NIH Black Swiss mouse eyes is reported by Aihara et al.¹² in the current issue. This procedure involves flattening the anterior chamber by aqueous aspiration followed by laser treatment of the limbus. IOP in these mice, normally approximately 16 mm Hg, becomes elevated to an average of 23 mm Hg and remains elevated for up to 12 weeks. Ocular side effects of this procedure were observed only occasionally, were of relatively minor significance, and were typically resolved spontaneously. The present study was undertaken to determine whether elevated IOP in the NIH Black Swiss mouse eye induces optic nerve axon loss and gliosis.

	Materials and Methods

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Animals
Animals used in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male NIH Black Swiss mice were obtained 6 weeks after birth from Taconic Laboratory (Germantown, NY) and 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-dark cycle. All mice were fed ad libitum. Two groups of mice were studied: one untreated group of 7 mice, to assess normal interindividual and intereye variability in the number of optic nerve axons and glial cells, and a second group of 13 laser-treated mice, to assess the relationship of induced ocular hypertension with optic nerve axon loss and glial proliferation.

Induction of Experimental Ocular Hypertension
The mice were anesthetized by 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). Aqueous outflow in the left eye was obstructed by laser photocoagulation at the limbus. The effect of this treatment was enhanced by flattening the anterior chamber before laser photocoagulation, as described by Aihara et al.¹² Animal age was 8 weeks at the time of laser treatment. The fellow eye served as a control.

IOP Measurements
IOP was measured in both eyes after laser treatment, every week in the first 4 weeks and every 2 weeks thereafter, as described by Aihara et al.¹³ Mice were anesthetized with an intraperitoneal injection of a solution containing ketamine (100 mg/kg) and xylazine (9 mg/kg). A fluid-filled glass microneedle connected to a pressure transducer was inserted through the cornea into the anterior chamber to measure IOP. All the mice completed this treatment regimen, with the exception of one mouse that died of unknown causes at 6 weeks.

The mean and maximum IOPs in the contralateral control and treated eyes and the duration of IOP elevation in the treated eyes were calculated. For each animal, a graph of IOP over time was constructed for the control and treated eyes. The area under the curve of control and treated eyes was calculated in units of mm Hg-weeks. The area under the control eye curve was subtracted from the area under the treated eye curve to assess the magnitude of IOP elevation. The total area for the period during which the IOP in the treated eye was higher than in the control eye, called the positive integral IOP, provided an estimation of the total exposure of the treated eye to elevated IOP.

Optic Nerve Axon and Glia Cell Counting
At the conclusion of the 12-week observation period, the mice were anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg, Nembutal; Abbott Laboratories, North Chicago, IL) and exsanguinated by perfusion with mammalian Ringer’s solution containing lidocaine hydrochloride (0.1 mg/mL, Xylocaine; Astra USA., Inc., Westborough, MA) and heparin sodium (500 U/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 carefully dissected and placed in this fixative overnight. The optic nerves from the animal that died at 6 weeks also were dissected and placed in 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 perpendicular to the long axis of the optic nerves on an ultramicrotome and placed on polyvinyl formal-coated (Formvar; SPI, West Chester, PA) slotted grids. These sections were obtained at approximately 300 µm posterior to the nerve’s emanation from the globe. Sections were counterstained with 1% uranyl acetate and Sato lead, and viewed by electron microscope (model 1200 EX; JEOL, Tokyo, Japan).

The number of axons in the mouse optic nerves was assessed according the method developed by Williams et al.¹⁴ with minor modifications. For each optic nerve cross section, electron micrographs were taken at low magnification (200x) to measure the cross-sectional area. Then a series of 20 micrographs were taken at high magnification (10,000x) in a square lattice pattern in the following positions within the optic nerve: center, four micrographs; midperiphery, eight micrographs; and peripheral margin, eight micrographs (Fig. 1) . No adjustments in position were made with respect to the tissues including blood vessels and glial cells. To confirm the true magnification, calibration grids were photographed at the same low (1000 mesh/in., no. 79525-01; EMS, Fort Washington, PA) and high (2160 lines/mm, no. 206; Ted Pella, Redding, CA) magnifications.

View larger version (10K): [in this window] [in a new window]   FIGURE 1. Relative position of micrographs obtained for analysis of axon density in mouse optic nerves. Oval circle indicates optic nerve cross section. Rectangular squares: positions in which micrographs were taken.

View larger version (109K): [in this window] [in a new window]   FIGURE 2. High-magnification (10,000x) images before (A) and after (B) analysis. A counting frame (7 x 8 µm) was traced on each image, and all survival myelinated and unmyelinated axons within the frame and intersecting the upper and left edges were marked and counted manually, according to standard unbiased counting rules. Each black dot indicates a counted axon.

To evaluate the reproducibility of the axon-counting method, three adjacent sections were analyzed from each of two different optic nerves. For each section, the position of the high-magnification micrographs was established according to measurements from the center of the optic nerve. Although corresponding images from successive sections were from similar regions, inspection confirmed that they did not overlap. The area of optic nerve cross section, the mean axon density, and the total number of axons were compared among the three sections from each nerve.

Statistical Analysis
Paired t-test and linear regression were used for evaluation of study results. P < 0.05 was considered to be statistically significant.

	Results

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Reproducibility of Axon Counting
The measurements of the area of optic nerve cross section, the mean axon density, within the three adjacent sections had coefficients of variation less than 3% (Table 1) . The measurements of total number of axons within the three adjacent sections had coefficients of variation less than 1.4%, which indicated that this method effectively identified differences in cross-sectional area or mean axon density that exceeded 3% or differences in the total number of axons that exceeded 1.5%.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. The mean and maximum intraocular pressure (IOP) in the control () and treated () eyes (n = 13). The mean IOP is the mean of all measurements during the observation period. Compared with the control eyes, the treated eyes had higher mean and maximum IOP (paired t-test). Bars, SE. Asterisks indicate P < 0.002.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. The optic nerve cross-sectional area (A), mean density of optic nerve axon (B), and total number of optic nerve axons (C) in the control () and treated () eyes (n = 13). Bars, SE.

View larger version (135K): [in this window] [in a new window]   FIGURE 5. Low- and high-magnification images of the optic nerve cross section in the control eye (A, C, E) and the treated eye with 99.5% axon loss (B, D, F) in M33 mouse. The area of optic nerve and mean axon density in the treated eye were 50% and 1% of the control eye, respectively. Most of the remaining myelinated axons had degenerated, and prominence of glial profiles was increased (F). Magnification: (A, B) x200; (C, D) x4,000; (E, F) x10,000.

View larger version (13K): [in this window] [in a new window]   FIGURE 6. There was a linear correlation between the decrease of optic nerve cross-sectional area and the loss of optic nerve axons (n = 13, r2 = 0.79, P < 0.0001, linear regression).

View larger version (13K): [in this window] [in a new window]   FIGURE 7. There was a linear correlation between the magnitude and duration of elevated intraocular pressure (IOP) and the loss of optic nerve axons (n = 12, r2 = 0.36, P = 0.040, linear regression).

View larger version (35K): [in this window] [in a new window]   FIGURE 8. The mean density of optic nerve astrocytes (A) and the number of astrocytes per optic nerve cross section (B) in the control () and treated () eyes (n = 12). Compared with the control eyes, the density and number of astrocytes were significantly greater in the treated eyes (paired t-test). Bars, SE.

View larger version (15K): [in this window] [in a new window]   FIGURE 9. There was a significant inverse linear correlation between the mean density of optic nerve axons and mean density of astrocytes in the treated eyes (n = 12, r2 = 0.38, P = 0.033, linear regression). In brief, the lower the axon density became, the higher the astrocyte density became.

	Discussion

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In human eyes, the optic nerve head is supplied by the arterial circle of Zinn and Haller formed by the choroidal arteries.^{17 18} In contrast, the arterial supply of the mouse optic nerve head derives from branches of the central retinal artery, and none of the capillaries are derived from choroidal vessels.¹⁹ A direct effect of IOP on optic nerve circulation in the mouse may be stronger than in humans. However, the IOP generated by this model was less than 40 mm Hg, whereas systolic blood pressure is greater than 100 mm Hg and diastolic blood pressure is near 70 mm Hg. Thus, IOP elevation was unlikely to be enough to occlude major retinal vessels. There was also no correlation between maximum IOP and axon loss. Although transient high IOP generally occurs after laser treatment of human eyes,²⁰ the IOP measured during the first 3 days after laser treatments of mouse eyes did not exceed 35 mm Hg.¹² Thus, it is unlikely that the axon loss in the present model is due primarily to optic nerve ischemia.

John et al.⁹ observed that DBA/2J mice with IOP elevation secondary to abnormalities in the anterior segment of the eye had retinal ganglion cell loss and optic nerve atrophy similar to that in glaucoma. Mice in the present model also had substantial axon loss. Moreover, the decrease in optic nerve cross-sectional area correlated with axon loss, as described in rat^{21 22} and monkey²³ eyes with experimentally elevated IOP and in human²⁴ glaucomatous eyes. In addition, optic nerve damage in the present model was related to the magnitude and duration of elevated IOP. Thus, the presently described mouse model mimics many features of chronic human glaucoma. However, the mechanism of optic nerve damage in this mouse model may be partially different from that in human glaucoma, because the mouse optic nerve head has no lamina cribrosa.¹⁹ The role of the lamina cribrosa in optic nerve damage in glaucoma is not clearly understood. Axonal transport is blocked in the region of the lamina cribrosa in monkey eyes with experimentally elevated IOP, which leads to axon loss in the optic nerve.^{25 26} The regional variation in lamina cribrosa anatomy is contrasted with susceptibility patterns of optic nerve head tissue to IOP elevation in eyes in a monkey glaucoma model.²⁷ Although the lamina cribrosa may play an important role in glaucomatous optic nerve damage, it is interesting that axon loss of the optic nerve is induced in mouse eyes by elevated IOP despite the absence of the lamina cribrosa. This raises the possibility that the IOP-induced changes in the lamina cribrosa may not be essential for IOP-induced optic nerve damage. Further investigations may clarify whether elevated IOP in the mouse eye disrupts axonal transport in the optic nerve.

Although there was a correlation between axonal loss and the IOP integral scores, it was not strong (r² = 0.36). In particular, four mice with integral scores ranging from 38 to 48 mm Hg-weeks had axon losses ranging from 10% to 99% (Fig. 7) . Of interest is that the duration of elevated IOP experienced by two of these mice that had axon losses less than 20% was 4 weeks only (M64 and M73, Table 3 ). In contrast, the duration of elevated IOP in the two mice with axon losses exceeding 95% was equal to or greater than 6 weeks (M31 and M109). Hence, consideration of the duration of elevated IOP, along with the integral score, may improve prediction of axon loss. It should be noted that when the integral score exceeded 48 mm Hg-weeks, axon loss always was greater than 80%. Hence, the duration of IOP elevation may be more important to consider when the integral score is less than 48 mm Hg-weeks and less important when the score exceeds 48 mm Hg-weeks. In a second example, mice M32 and M34 had different axon loss (52% and 99.8%, respectively), although IOP was similarly elevated for 6 weeks, and they had the same value for positive integral IOP. However, mouse M34 had a period of 6 weeks of normotension after the time of elevated IOP, whereas mouse M32 did not. This may be consistent with the axon loss in rat optic nerves that experienced a period of normal IOP that followed a 3-week period of significant IOP elevation.²¹ Hence, considering the duration of elevated IOP or total time since laser treatment, as well as the integral IOP score may improve prediction of axon loss. Further supporting the link of pressure elevation to axon loss is the observation of no axon loss in one treated eye in which IOP never exceeded 21 mm Hg (M113). Moreover, this eye had undergone transient hypotony, as did each of the treated eyes, suggesting that transient hypotony does not cause axon loss in this model.

Several other possible sources of variation in axon loss may exist. These include regional differences in axon density within the nerve, genetic variation among individual mice,^{14 28 29} and possible effects of environmental factors.¹⁴ In addition, small intereye variations were noted. These are likely to represent real differences, because they exceed by threefold the variations in repeat counts of the same optic nerve. Similar individual and intereye variations have been noted in human optic nerves.^{30 31} These latter sources of variation, though normal, may limit the ability to use the contralateral eye as a control for the optic nerve axon counts in the treated eye.

Astrocytes are the major glial cell type in the optic nerve in most mammalian species, and mature, quiescent astrocytes are reactivated and participate in formation of a glial scar in glaucomatous optic neuropathy.³² As to the proliferation of astrocytes, although there is a pool of astrocytes capable of entering the mitotic cycle, their proliferative capacity appears to be limited in the adult human brain.³³ Quigley and Anderson³⁴ reported that no evidence of astrocyte mitosis was observed after optic nerve transection in the monkey, and the estimated volume of astrocytes increased only slightly from normal. Furuyoshi et al.³⁵ also described an increase in glial density, accompanied by a decrease in optic nerve cross-sectional area, whereas the total number of astrocytes remained nearly constant in a monkey glaucoma model. However, in the present mouse model, not only the density of astrocytes, but also the total number of astrocytes per optic nerve cross section increased, which suggests proliferation of astrocytes. This different response of astrocytes may depend on species, because there is undeniable evidence for astrocyte proliferation in response to brain injury in the mouse.³⁶ Kwiecien et al.³⁷ also reported glial proliferation in the optic nerve in demyelinating mutant rats. The proliferation of astrocytes in the present mouse model may be induced by a similar mechanism, because demyelination happens in association with the process of axon loss. The increase in number of astrocytes observed in the present model may indicate that they have been reactivated.

In conclusion, the present study confirms a positive correlation between experimental elevated IOP in the mouse eye and associated loss of optic nerve axons. Similarities between this model system and human glaucoma suggest that this model may be useful for evaluating the cellular mechanisms of glaucoma as well as the potential of new treatments for glaucoma.

	Acknowledgements

 
The authors thank Simon John, Jackson Laboratories (Bar Harbor, ME), for a critical reading of the manuscript.

	Footnotes

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

Submitted for publication February 7, 2003; revised May 13 and July 11, 2003; accepted June 14, 2003.

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