Abstract
Purpose.:
Intraocular pressure (IOP) fluctuations may occur in patients with glaucoma, but how these fluctuations affect axonal populations in the optic nerve and other structures in the eye has been difficult to assess. This study developed a rat model to evaluate the effect of intermittent controlled elevations in IOP on the morphology of the rat optic nerve.
Methods.:
IOP was transiently elevated for 1 hour on each of 6 days a week over 6 weeks with an adjustable vascular loop around the right topically anesthetized eye of Sprague-Dawley rats. IOP was measured by pneumatonometer before, immediately after, and at the end of 1 hour of treatment with ligature. Globes and optic nerve segments were prepared for histology and morphometry.
Results.:
Mean baseline IOP of 14.9 ± 1.8 mm Hg increased to 35.3 ± 2.6 mm Hg (P < 0.001) during 1-hour treatments and returned to 15.0 ± 2.2 mm Hg (P = 0.84) 1 hour after completion. The contralateral untreated eyes had a mean IOP of 14.2 ± 1.9 mm Hg at baseline and 14.6 ± 1.9 mm Hg at the end of treatment. Nerve fiber layer thinning (22%–25%) corresponded with a decrease (7%–10%) in soma number in the ganglion cell layer. Optic nerves displayed axonal degeneration with a modest axon loss of 6% and increased expression of glial acidic fibrillary protein in astrocytes.
Conclusions.:
Controlled daily 1-hour IOP elevations can be performed with an adjustable vascular loop in rats. After only 6 weeks, intermittent elevations in IOP produce changes in optic nerve consistent with early degeneration reported in chronic models of glaucoma.
Chronically elevated intraocular pressure (IOP) is a well-known and well-studied risk factor for glaucomatous optic neuropathy. However, some patients with successful treatment to lower IOP as measured in the clinic, or others treated for normal pressure glaucoma, may have continued glaucomatous progression. This has been hypothesized to be related to transiently elevated IOP perhaps because of medical noncompliance,
1,2 medication troughs,
3 an inversion, Valsalva, or other activities increasing IOP,
4,5 or diurnal fluctuation.
6 –11 The detrimental effects of intermittent moderate elevations of IOP on the optic nerve and retina are observed in patients with intermittent uveitic glaucoma
12 or in those with IOP elevations from intermittent steroid use.
13 Furthermore, IOP fluctuations have been suggested to be more common in patients with glaucoma than in subjects without it.
7,14 Greater diurnal IOP fluctuation was recently found in patients treated medically rather than surgically.
15 In a prospective study of 76 patients, those with the largest range of measured IOP had the greatest visual field deterioration over a 2-year period.
16 Similarly, a consistent IOP <18 mm Hg resulted in no visual field deterioration, whereas achieving <18 mm Hg in <50% of visits resulted in visual field deterioration in the Advanced Glaucoma Intervention Study.
17 Therefore, investigating IOP fluctuations in an animal model is clinically relevant.
Current genetic and surgically induced chronically elevated IOP animal models are noted for their variability in IOP measurements among animals.
18 –32 However, it is difficult to standardize and explore these fluctuations in a controlled investigation in these models. A well-studied controlled IOP elevation model is used to investigate ocular ischemia.
33 –35 However, this rat model represents an extreme global ischemia with IOP elevation beyond the ocular perfusion pressure. In this model, 20 minutes of IOP elevation will produce irreversible ERG changes,
33 and 45 minutes will produce histologic changes.
34 This absolute global ocular ischemia is not representative of the conditions associated with the majority of glaucomas.
35 In addition, this model requires systemic anesthesia and invasive cannulation of the eye, which would be difficult to repeat multiple times in one animal.
The effects of intermittent IOP elevation are not well studied because of lack of control over such fluctuations. A controlled nonsurgical model may offer insights into the earliest changes that could occur in the eye after specified IOP elevation. A model in which IOP can be elevated to partially damage the retinal ganglion cells (RGCs) and their axons and then be restored to normal levels is also desired by investigators to understand which functions are not restored in partially damaged RGCs. This information may suggest potential new therapies to treat injured cells.
36 The purpose of this study was to develop a model in which multiple controlled IOP elevations can be consistently produced in a relatively noninvasive manner and to determine whether basic glaucomatous histologic alterations are detectable after 6 weeks of daily 1-hour IOP elevation.
Rats were given a lethal dose of anesthesia intraperitoneally and were perfused transcardially with heparinized phosphate-buffered saline followed by 3% paraformaldehyde (catalog no. 158127; Sigma-Aldrich, St. Louis, MO), 0.1% glutaraldehyde (catalog no. 16220; Electron Microscopy Sciences, Hatfield, PA) (vol/vol), and 0.2% saturated picric acid (catalog no. LC18670; LabChem Inc., Pittsburgh, PA) (vol/vol) in 0.1 M phosphate buffer. After perfusion, the globes and optic nerves were enucleated and separated. The globes were postfixed overnight in the perfusion fixative, dehydrated in graded alcohols and acetone, and embedded in paraffin. Sagittal 7-μm serial sections were stained with hematoxylin (SL90; Statlab, Lewisville, TX) and eosin (C.I. 45380; EMS, Hatfield, PA) for light microscopy.
A masked observer photographed 40× fields (AX70; Olympus, Melville, NY) from serial sections of treated and untreated eyes. Photographs were taken 1 to 2 mm from the optic nerve and in the periphery. The nerve fiber layer (NFL) thickness was measured in three equidistant regions across each photograph using image analysis software (Image Pro Plus 5.1; Media Cybernetics, Silver Spring, MD).
38 Comparisons were made between untreated and treated eyes for both central and peripheral measurements. Cells, including RGCs and displaced amacrine cells, within the ganglion cell layer also were counted within the entire 40× fields with this software.
39 Again, comparisons were made between untreated and treated eyes for both central and peripheral measurements.
The optic nerves 2.5 mm proximal to the globes were removed and fixed in 2% glutaraldehyde (Electron Microscopy Sciences). They were postfixed in 1% osmium tetroxide (catalog no. 19100; Electron Microscopy Sciences) in cacodylate buffer for 1 hour, dehydrated in graded ethanol and acetone, and embedded in Epon (Embed 812; Electron Microscopy Sciences) for evaluation of whole nerve and individual axons. Semithin 1- to 2-μm sections were stained with toluidine blue (Electron Microscopy Sciences) for morphologic evaluation. Ultrathin sections were cut from selected specimens, collected on 200 mesh grids, stained with uranyl acetate (Electron Microscopy Sciences) and lead citrate (Electron Microscopy Sciences), and viewed under a transmission electron microscope (CM-12; Phillips, Hillsboro, OR).
Axon counts were obtained as previously described.
40,41 Briefly, cross-sections of optic nerve were photographed en montage at 100× magnification (AX70; Olympus, Melville, NY). A previously described set of custom algorithms (courtesy of David Calkins, Vanderbilt University, Nashville, TN) was used to identify and count axons across the entire cross-section as well as measure the entire cross-sectional area of the nerve.
40 This algorithm identifies axons that are defined by the presence of a myelin sheath and counts all these axons in each individual 100× image for the entire montage of the nerve. The resultant data are an actual count of all axons with a myelin sheath that are visible at 100× in the optic nerve cross-section. To control for the previously described variability in axon number between individuals,
42 axon data are represented as the ratio of total axon counts in nerves from treated and untreated fellow eyes. Degenerative profiles were quantified by hand counting of axons with swelling or axonal debris in semithin sections. These counts were performed by a trained, masked observer and are represented as the ratio of degenerative profiles in nerves from treated and untreated fellow eyes. Degenerative changes in the ultrastructure of axons were examined in electron micrographs of nerve cross-sections.
Degeneration of the optic nerve also includes activation of glial cells, including astrocytes. Changes in cytoskeletal elements are a hallmark of astrocyte reactivity. To specifically evaluate the reactivity of astrocytes in the optic nerve, we examined changes in immunolabeling of the intermediate filament and astrocyte marker glial fibrillary acidic protein (GFAP) in longitudinal sections of optic nerve. Paraffin sections were deparaffinized and treated for 6 minutes in proteinase K antigen retrieval buffer (S3020; DakoCytomation, Glostrup, Denmark). Sections were then blocked with 5% goat serum and 1% BSA for 30 minutes and subsequently were incubated in primary antibody GFAP (1:1000; Z0334; DakoCytomation) at 4°C overnight. Secondary staining was performed with AlexaFluor 488 F(ab)2 fragment of goat anti-rabbit IgG (H+L) at 1:100 concentration (A11019; Invitrogen, Carlsbad, CA). Controls for immunohistochemistry experiments were conducted with no primary antibody and the appropriate IgG isotype. At least six confocal micrographs (60× magnification) were obtained for each nerve analyzed on an upright confocal microscope equipped with laser scanning fluorescence (blue/green, green/red, red/far-red) and Nomarski-DIC, 3D z-series, and time series (LSM 510; Zeiss, Thornwood, NY) in the Vanderbilt Cell Imaging Core. Changes in immunolabeling intensity were quantified by spectral analysis of fluorescent signal using an image analysis software package (Image Pro Plus; Media Cybernetics, Silver Spring, MD).
IOP Measurements.
Retinal Pathology.
The Mann-Whitney rank sum test was used to compare the thickness of the nerve fiber layer in untreated versus treated retinas. A two-sample, unpaired t-test was used to compare the number of cell soma in the ganglion cell layer in untreated versus treated retinas. To determine the appropriate statistical tests, normality (Shapiro-Wilk Normality Test) and variance (Equal Variance Test) of all data were examined before variance analysis. All analyses were conducted in statistical analysis software (SigmaPlot/SigmaStat, version 11.0; Systat, Inc., San Jose, CA). For all analyses, P ≤ 0.05 was considered statistically significant.
Optic Nerve Pathology.
The Wilcoxon signed rank test was performed to compare the numbers of degenerating axons per cross-section between the two eyes. A one-sample t-test was performed to compare the ratios of total axons, cross-sectional area, and GFAP intensity in treated and untreated optic nerves to the hypothetical population value of 1.0. Before variance testing, the normality of all data was examined using the Shapiro-Wilk normality test. All analyses were conducted in statistical analysis software (SigmaPlot/SigmaStat, version 11.0; Systat, Inc.). For all analyses, P ≤ 0.05 was considered statistically significant.
The presented model provides an opportunity to investigate the effects of intermittent elevation of IOP on the rat eye. Targeted IOP elevations are possible with the adjustable ligature. It may be easily tightened or loosened to achieve the desired IOP. Daily IOP elevations for 1 hour are possible with topical anesthesia in this animal model without systemic stress at least up to 6 weeks. This is an advantage over the confounding variables of systemic anesthesia and surgical manipulation to raise IOP when it is desired to evaluate the earliest effects of elevated IOP. Others have clearly documented the detrimental effects such as weight loss with frequent systemic anesthesia administration to measure the IOP in rats.
51 As can be seen in
Figure 3, the effect of the lasso was substantial for the IOP in the treated eye, whereas the untreated eye underwent statistical but little clinically relevant change in IOP. This elevation was sustainable over the 1-hour treatment period with only a slight decrement.
Unlike the genetic model of the DBA/2J mouse,
25 the angles in the proposed model appear to remain open without synechiae. The development of synechiae would be expected to produce chronic elevations of IOP. That IOP returned to baseline levels even at the end of the 6-week trial further supports the lack of synechiae in this model. Unlike other models,
22,25,29 inflammation was not noted in the anterior chamber (
Fig. 4). Histologic examination of retinal sections also revealed that the retinal layers remained intact (
Fig. 4). This is in contrast to that observed after acute ischemia-reperfusion manipulations in which the IOP is elevated above perfusion pressure for only 1 hour and is followed over time.
34 Overall, our data suggest that the vascular loop elevates IOP without significant ischemic damage.
The shortest period of moderate IOP elevation required to produce degenerative changes in the retina and optic nerve is unknown. Here we describe various indicators of degeneration in the ganglion cell and nerve fiber layers of the retina and optic nerve after only 1 hour of elevated IOP daily for 6 weeks. In the retina, we demonstrated that intermittent IOP elevation to 35 mm Hg induced both a reduction in NFL thickness and a decrease in the number of cell soma in the ganglion cell layer that, though present, is lower than that reported in an advanced glaucoma model (
Fig. 5).
52 Intermittent IOP elevations had a greater impact on NFL thickness (22%–25%) than the number of cell soma in the ganglion cell layer (7%–10%). This is likely because of the presence of displaced amacrine cells, whose processes are not a component of the NFL, in the ganglion cell layer.
Our qualitative analysis of optic nerve by both light and electron microscopy revealed an increase in the number of degenerative axon profiles and vacuolization in the extra-axonal space (
Fig. 6). At the level of individual axons, we demonstrated evidence of disorganized myelin sheaths, disruption of neurofilaments, and enlargement of the axoplasmic space (
Fig. 6). These axonal changes are consistent with optic nerve axon degeneration well described in other studies.
21,25,30,53 –55 Interestingly, our quantitative analysis of degenerative axon profiles revealed significant variability among treated eyes despite almost identical IOP elevation. Although it is possible that the small variation in age (2 months) among our subjects could contribute to variability between individuals, it is also possible that these differences represent intrinsic variability that is dependent on a variety of factors, including genetic differences. It is intriguing that the highly uniform nature of IOP elevation in this model may provide a means to investigate responses to elevated IOP at the level of the individual. It is important to note that though there was individual variability in the number of degenerative profiles among treated eyes, all treated eyes qualitatively demonstrated some indicators of degeneration (e.g., increase in axoplasmic space, disruption of neurofilaments) compared with their contralateral untreated eye. In any biological system, including the optic nerve, the rate and features of the degenerative process are not uniform across cells exposed to the same stimulus. This is well demonstrated in vitro, when exposure to elevated hydrostatic pressure is identical for all cells on the culture plate but induces apoptosis in RGCs that increases over time rather than occurring all at once.
56
In accordance with our retinal analyses, quantitative analysis of total axon number revealed a decrease of 6% in optic nerves from treated eyes versus the contralateral untreated eye (
Table 1;
Fig. 7). This decrease in axon number appeared to be diffuse rather than focal across the optic nerve. Over longer periods of exposure, this diffuse degeneration could remain diffuse over time,
40 evolve into primarily focal axonal lesions,
19 or evolve into a combination of diffuse to focal defects.
57
Published reports of axon counts in rat optic nerve vary depending on several factors, including age extremes of the rat
42,58,59 and the sampling techniques used.
23,42,58 –60 Our average of 92,919 ± 13,442 axons per untreated nerve compares favorably with the approximate 96,200 to 108,100 axons in albino rats, as described by time-intensive transmission electron microscopy sampling techniques.
58,60 Cepurna et al.
42 found more axons (117,900 ± 11,000) in the Brown Norway optic nerve with electron microscopy. Although the smallest axons may be missed in quantitative whole nerve counts at the light microscopic level, their presence or absence would also be overlooked in the alternative qualitative grading systems.
32,61 Furthermore, when performing axon counts, it must be considered that there is biological variability among animals and between eyes in the same animal. Cepurna et al.
42 reported up to 8.5% variability of optic nerve axon counts between the right and left eye of an animal with a much greater variability among eyes of different 5-month-old Brown Norway rats.
42 To compensate for the variability among animals, axon counts were evaluated as treated/untreated ratio for each animal. In support of our findings in the optic nerve, the magnitude of axon loss we detected (6%) 2.5 mm from the globe was strikingly similar to that of cell soma loss in the ganglion cell layer (7%–10%).
In our quantitative analyses of optic nerve, we also discovered a tendency toward shrinkage of the optic nerve. However, this trend was not significant (
Fig. 7). It is possible that this tendency for smaller optic nerves in treated eyes could result from a loss of axons and, therefore, an increase in severity over time. We cannot rule out the possibility that this trend could also be attributed to histologic shrinkage of the tissue. This is particularly true given the variability across animals.
In addition to our analysis of RGC degeneration, we also examined the impact of intermittent IOP elevations on the reactivity of astrocytes in the optic nerve. In central nervous system injury and human glaucoma, hypertrophy and upregulation of GFAP in astrocytes are components of the glial cell response to neurodegeneration.
46 –50 For animal glaucoma models with continuous elevations of IOP, the timeline of astrocyte reactivity varies.
21,62 –64 In advanced glaucomatous damage, elevated GFAP was prevalent in the optic nerves in animal models.
63,64 The effect of intermittent IOP on glial cell reactivity has not been examined previously. In this model, the upregulation of GFAP was evident in optic nerves from treated eyes compared with the contralateral untreated eyes (
Fig. 8). As in our analysis of degenerative profiles in the optic nerve, there was significant variability among treated eyes. Interestingly, the magnitude of GFAP reactivity correlated with the number of degenerative profiles; the two animals (103 and 104) with the smallest ratios of degenerative profiles also had modest increases in GFAP intensity (compare
Fig. 6 and
Fig. 8). Furthermore, these two animals had total axon counts that were close to the mean for treated eyes. These data suggest that though the irreversible outcome of the disease process (i.e., axon loss) was similar among treated animals, the process by which this outcome is achieved may vary inherently between individuals with nearly identical insults.
In summary, consistent intermittent IOP elevations can be produced daily for 1 hour in the adult rat without evidence of overt ischemia or stress to the animal. Six weeks of daily moderate IOP elevations will produce early histologic glaucomatous damage with modest degeneration of RGCs and their axons that share characteristics with degeneration induced by continual elevations of IOP. This model provides an opportunity with which to evaluate early molecular and histologic responses to IOP fluctuations that are increasingly believed to contribute to glaucomatous progression in a controlled and reproducible manner. In addition, our data suggest that the highly controlled nature of these IOP elevations provides the potential to examine individual variability in the IOP-induced degenerative process without confounding factors, such as rate and magnitude of IOP elevation. This knowledge may assist in developing therapies to effectively treat injured retinal ganglion cells and their axons in an environment of fluctuating IOP.
Supported by Fight for Sight Grant-in Aid GA05035 (KMJ); Joseph Ellis Family Glaucoma Research Fund (KMJ); Vanderbilt CTSA voucher from National Center for Research Resources/National Institutes of Health Grant UL1 RR024975 (KMJ); National Institutes of Health Core Grant in Vision Research Grant 2P30EY008126–21; and an unrestricted departmental grant from Research to Prevent Blindness, Inc.
Disclosure:
K. Joos, None;
C. Li, None;
R. Sappington, None
The authors thank Peter Haddix, Anta'Sha Jones, Lauren Knish, Ratna Prasad, Meena Putatunda, Richard Robinson, Sanaz Saadat, Stephanie Sims, and Guangming Wu for valuable technical assistance; David Calkins for algorithms for the optic nerve analysis; and the staff of the Cell Imaging Core at Vanderbilt University Medical Center for aid in obtaining micrographs.