Axonal Transport and Cytoskeletal Changes in the Laminar Regions after Elevated Intraocular Pressure

Abstract

purpose. To investigate the axonal cytoskeleton changes occurring in the prelaminar region, lamina cribrosa, and postlaminar region of the porcine optic nerve after an acute increase in intraocular pressure (IOP) and whether this corresponds with axonal transport abnormalities.

methods. Six white Landrace pigs were used. The left eye IOP was elevated to 40 to 45 mm Hg for 6 hours, and the right eye IOP was maintained between 10 and 15 mm Hg. Rhodamine-β-isothiocyanate (RITC) was injected into the vitreous of each eye at the beginning of the experiment, to study axonal transport. After euthanasia, optic nerves were removed and prepared for axonal transport and cytoskeleton studies. Antibodies to phosphorylated neurofilament heavy (NFHp), phosphorylation-independent neurofilament heavy (NFH), neurofilament light (NFL), neurofilament medium (NFM), microtubule, and microtubule-associated protein (MAP) were used to study the axonal cytoskeleton. Montages of confocal microscopy images were quantitatively analyzed to investigate simultaneous changes in optic nerve axonal transport and cytoskeletal proteins in the high-IOP and control eyes.

results. Axonal transport of RITC was reduced in the prelaminar, lamina cribrosa, and proximal 400 μm of the postlaminar optic nerve regions in the high-IOP eye. NFHp, NFM, and NFH were significantly reduced in the prelaminar, lamina cribrosa, and proximal postlaminar regions in the high-IOP eye. No differences in NFL, MAP, and tubulin staining were detected.

conclusions. Elevated IOP induced both axonal transport and cytoskeleton changes in the optic nerve head. Changes to the cytoskeleton may contribute to the axonal transport abnormalities that occur in elevated IOP.

Increased intraocular pressure (IOP) is a major risk factor predictive of visual loss due to retinal ganglion cell (RGC) damage in glaucoma, ¹ the second leading cause of blindness worldwide. ² The mechanism underlying RGC loss in glaucoma has not been clearly elucidated. An increased pressure gradient across the lamina cribrosa, resulting in posterior displacement and deformation of this structure, has been proposed. ³ This increased pressure gradient may cause direct compression of RGC axons and changes in blood flow resulting in modifications within the axonal microarchitecture and disturbances in axonal transport. ⁴  

The axon plays a crucial role in connecting neurons and acting as a conduit for the transmission of information between them. Axonal transport is vital for maintaining the trophic effect on the neuron and the tissues innervated by that neuron. ⁵ The human RGC axon travels a distance of approximately 50 mm from the cell body to its target synapse. Obstruction of axonal transport in the RGC compromises the viability of the cell by preventing the arrival of substrates, such as neurotrophic factors, that are necessary for somal survival. ⁶ Failure of this transport results in apoptotic cell death. ⁷ The axonal cytoskeleton forms the molecular tracks for the bidirectional movement of motor proteins and their associated cargo in addition to maintaining RGC and axon shape. ⁸ Neurofilaments, microtubules, and microtubule-associated proteins (MAPs) are major constituents of the axonal cytoskeleton. Neurofilaments are heteropolymeric structures composed of three individual subunits: neurofilament heavy (NFH), neurofilament medium (NFM), and neurofilament light (NFL), in order of decreasing mass. All three subunits are composed of a highly conserved α-helical central coiled rod domain flanked by an N terminus head domain and a C terminus hypervariable tail domain. ⁹ Structurally, neurofilaments are linearly arranged and composed of a helical filament backbone with sidearms that project laterally to form crossbridges. ¹⁰ The NFL subunit forms the core of these filaments, whereas the unique hypervariable C terminus tail domains of NFM and NFH, enriched in Lys-Ser-Pro repeat motifs are responsible for forming the neurofilament sidearms. ⁹ Phosphorylation of these sidearms is important in regulating the degree of interaction between neurofilaments and other axonal structures. ¹¹  

Disruption of axonal transport and cytoskeletal damage with associated pathogenic factors have been reported in the mouse model of optic nerve stretch injury. ¹² Although high-IOP-induced changes in axonal transport ^{4 13 14} and axonal cytoskeletal abnormalities ¹⁵ have been reported in independent reports, we are unaware of previous studies examining IOP-induced changes in axonal transport concurrently with axonal cytoskeleton changes in the optic nerve head. We were interested in exploring the earliest cytoskeletal changes that occur with an acute increase in IOP. An acute experimental protocol was used, as it allowed us to control and monitor IOP, cerebrospinal fluid pressure (CSFp), and blood pressure closely. These parameters are difficult to control in a model of chronic glaucoma. The purpose of this study was to examine axonal transport changes, axonal cytoskeleton alteration, and their relationship in the prelaminar, lamina cribrosa, and postlaminar regions of the optic nerve after acutely raised IOP. We examined the distribution of RITC within optic nerve axons after intravitreal injection and performed immunohistochemical stains of cytoskeletal subunits on the same tissue. 

Materials and Methods

General

The experimental techniques were similar to our earlier report. ¹⁶ Six white Landrace female pigs weighing between 18 and 23 kg were used. Sedation was induced by an intramuscular injection of tiletamine-zolazepam 4.4 mg/kg (Zoletil; Virbac, Peakhurst, NSW, Australia) and xylazine 2.2 mg/kg (Xylazil; Troy Laboratories, Smithfield, NSW, Australia) and maintained with isoflurane (Isorrane; Baxter Healthcare Pty. Ltd., Old Toongabbie, NSW, Australia) in oxygen-nitrogen and an intravenous infusion of fentanyl citrate (Mayne Pharma Pty. Ltd., Mulgrave, VIC, Australia) at a rate sufficient to maintain anesthesia throughout the experiment. The femoral artery was catheterized to allow continuous measurement of blood pressure and intermittent collection of arterial blood samples for arterial blood gas analysis (Rapidlab 248; Bayer Corp., Medfield, MA). Ventilator settings were adjusted accordingly to maintain blood pH, oxygen, and carbon dioxide tensions within the normal range. An infusion of isotonic fluid (Hartmann’s; Baxter Healthcare Pty. Ltd.) and dobutamine (Mayne Pharma Pty. Ltd., Mulgrave, VIC, Australia) were titrated to maintain blood pressure within the normal range. Pancuronium (AstraZeneca, North Ryde, NSW, Australia) was also given to permit muscle relaxation. 

Stereotaxic coordinates were used to place two cannulae into the lateral ventricles of the brain. One cannula was attached to a variable height infusion of Ringer’s lactate to control CSF pressure, and the contralateral side connected to a pressure transducer. After lateral canthotomy, an eye ring was sutured to the perilimbal sclera of each eye. Then, two 25-gauge cannulae inserted to its anterior chamber and connected to a pressure transducer and a variable-height infusion of Ringer’s lactate, so that IOP could be monitored and adjusted. The right eye was used as the control eye, with the IOP maintained between 10 and 15 mm Hg, whereas the left eye was designed as a high-IOP model, with the IOP being between 40 and 45 mm Hg for 6 hours. The duration and IOP settings for this study were based on previous report involving primates ¹⁷ and our preliminary work in pigs that also showed a reduction in axonal transport after 6 hours of elevated IOP at 40 to 45 mm Hg. 

The pupil was dilated, and the retina and optic disc was observed with an operating microscope (Carl Zeiss OPMI, Oberkochen, Germany). A 30-gauge needle was passed through the temporal sclera into the vitreous, and 100 μL of freshly prepared 3% RITC ¹⁸ was injected into the vitreous of each eye for the study of axonal transport. After injection both eyes were covered with black rubberized plastic (Thorlabs Inc., Newton, NJ) to prevent bleaching of the fluorescent tracer. 

All pressure transducers (P23 ID Gould Statham; Gould, Cleveland, OH) were calibrated before the experiment and held at eye level. They were connected via a conditioning module (Analog Devices, Norwood, MA) to a chart recorder (model LR8100; Yokogawa, Tokyo, Japan) to allow continuous recording of IOP, CSFp, and blood pressure. Pigs were euthanized 6 hours after RITC injection with an anesthetic overdose of intravenous pentobarbitone (Lethabarb; Virbac Pty. Ltd., Peakhurst, NSW, Australia). All experiments were conducted and all laboratory animals treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the University of Western Australia Animal Ethics Committee. 

Tissue Preparation and Immunohistochemistry

Tissue preparation and immunohistochemistry protocols were adopted from those previously reported. ¹⁹ Eyes were immediately enucleated after euthanasia. The tissue was then carefully dissected to ensure complete removal of the dura from the optic nerve. The retina and the proximal 8 mm of the optic nerve was then carefully exposed. Dissected tissue was mounted in OCT and longitudinally sectioned into 12-μm specimens on a cryotome set at −30°C. To avoid the potential tilting of sections, we aligned the optic nerve parallel to the blade on the cryostat. Because the dura had been removed before sectioning, the optic nerve was clearly defined, and we were thus able to align the tissue accurately and ensure that the sections cut were horizontal with no tilting. In all pigs, optic nerves from both eyes were identically oriented before cryosectioning to allow accurate comparisons between prelaminar, lamina cribrosa, and postlaminar regions. Longitudinal sections were cut along the horizontal plane beginning in the superior portion of each optic nerve and proceeding to the inferior part of the nerve. For both axonal transport and cytoskeleton antibody studies sections from the central region of each optic nerve were used (Fig. 1) . By chronologically numbering each section that was cut from the superior to the inferior portion of the optic nerve, we were able to ensure that the levels of the sections used for comparison between the control and high-IOP eyes were reliably matched. 

Specimens to be used for the study of axonal transport were stored in the dark at −80°C immediately after sectioning and viewed the following day after mounting in glycerol. Some sections were stained with Van Gieson stain immediately after cryosectioning to determine prelaminar and lamina cribrosa thickness. Remaining slides were washed for 5 minutes in a wash solution composed of 0.01 M PBS and Tween 20 (Sigma-Aldrich, St. Louis, MO) before incubation with their primary antibody solution. Tissues from the control and high-IOP eye were incubated with antibodies prepared simultaneously from the same batch of reagents. 

All primary antibodies were made into solution with 1% goat serum (G9023; Sigma-Aldrich) and 1% bovine serum albumin. Triton X-100 (0.1%; Sigma-Aldrich) was also used for all primary antibody incubations, to improve permeability. Primary antibodies used in the present study were polyclonal antibody NF-L directed against the NFL subunit (1:500, AHP286, Serotec, Oxford, UK), monoclonal antibody NF-M directed against the NFM subunit (1:200; N5264, clone NN18; Sigma-Aldrich), monoclonal antibody NF-H directed against the phosphorylated and nonphosphorylated NFH subunit (1:400; N0142, clone N52; Sigma-Aldrich), monoclonal antibody NF-Hp directed against the NFHp subunit (1:200; N5389, clone NE14; Sigma-Aldrich), monoclonal antibody Tub directed against isotypes I and II of β-tubulin (1:200; T8535, clone JDR.3B8; Sigma-Aldrich) and anti-MAP antibody (1:200, M7273; Sigma-Aldrich). All primary antibodies were incubated for 24 hours, except for anti-NFL and anti-MAP, which were incubated for 48 hours. 

After primary antibody incubation, all specimens were given four washes over 20 minutes in a 4°C wash solution. Slides were then incubated at room temperature with a secondary antibody for 6 hours. Secondary antibodies included goat anti-mouse IgG (1:400; Alexa Fluor 488, A11001; Invitrogen-Molecular Probes, Eugene, OR) or goat anti-rabbit IgG (1:400; Alexa Fluor 488, A11008; Invitrogen-Molecular Probes). 

After secondary antibody incubation all specimens were washed four times over 20 minutes in a 4°C wash solution. Slides were then mounted in glycerol and immediately viewed with the confocal microscope. 

Image Acquisition

Digital images of labeled tissue were captured with a UV confocal laser scanning microscope (model 1000/1024 MRC controlled by COMOS image-acquisition software; Bio-Rad, Hercules, CA). Visualization of sections labeled with cytoskeletal antibodies was achieved by laser excitation at a 488-nm line from an argon laser with emissions detected through a 522/35 nm band-pass filter. Visualization of RITC-labeled tissue was achieved by laser excitation at a 543-nm line from a green helium neon laser with emissions detected through a 580/32-nm band-pass filter. An oil-immersion lens (PlanoApo 60× NA 1.4; Nikon, Tokyo, Japan) was used to view slides labeled with cytoskeletal antibodies, whereas a dry lens (20× NA 0.4; Nikon) was used to view RITC-labeled tissue. Images of the control and high-pressure eye labeled with the same primary antibody were acquired immediately after each other, using identical instrument settings being sure to avoid saturation of the brightest pixels. 

Using a motorized stage and a macro written in the confocal microscope software (MPL COMOS; Bio-Rad) a series of z-stacked images were captured of each slide, beginning in the prelaminar region and extending through to the postlaminar tissue. Images were collected in gray scale (on a scale of 0–255). Each z-stack consisted of a depth of optical sections collected at 2-μm increments along the z-plane. Each z-stack consisted of 7 sequential images collected using Kalman averaging. z-Stacks were then montaged by using a plugin in Image J (ver. 1.36; http:/rsb.info.nih.gov/ij/ developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Creating a montage allowed us to study spatial changes in the prelaminar, lamina cribrosa, and postlaminar regions of the optic nerve in detail. 

Quantitation of Prelaminar and Lamina Cribrosa Thickness

Van Gieson-stained slides were digitized with a high-resolution digital camera (DXM 1200; Nikon) attached to a microscope (Eclipse E80; Nikon). The digitized images were analyzed on computer (Image Pro Plus, ver. 5.1; Media Cybernetics, Silver Spring, MD) to isolate the lamina cribrosa region. The lamina cribrosa was identified by transverse arrangement of laminar beams. The anterior and posterior limits of the transverse beams were identified (Fig. 2) . Tissue bound by these anterior and posterior limits and by the scleral canal on each side was considered laminar tissue. The distance between anterior and posterior limits was taken as the thickness of the lamina. The distance from the inner limiting membrane to the anterior limit of the lamina was taken as the thickness of the prelaminar region. For lamina cribrosa thickness, three measurements within each region were taken: the temporal, adjacent to the temporal scleral canal; central; and nasal, adjacent to the nasal scleral canal. 

Quantitation of Cytoskeletal and Axonal Transport Differences

Quantitation of all images was performed on computer (Image Pro Plus, ver. 5.1; Media Cybernetics). 

Cytoskeleton.

For quantitating cytoskeletal change, each montaged z-stack was first deconstructed in gray scale into its original seven slices. Each of these slices was then analyzed separately. We therefore analyzed seven sections for each antibody in each eye (giving a group total of 42 pairs of sections per antibody). All images were randomized, such that the observer was masked to control and high-IOP images as well as the cytoskeletal antibody being analyzed. For each analysis the image was divided into three regions: prelaminar, lamina cribrosa, and postlaminar. The entire prelaminar and lamina cribrosa regions and the proximal 200 μm of the postlaminar region were analyzed. With a quantitative histogram function, the average pixel intensity per 1 μm² in each of the prelaminar, laminar, and postlaminar regions of each image was calculated by using a sample window of a constant size and sampling an equal number of random points. The mean pixel intensity in each region was then used for statistical analysis. Only neural tissue from the prelaminar, lamina cribrosa, and postlaminar regions was sampled. 

Axonal Transport.

For axonal transport analysis, the average intensity of the z-stack projection of each montage was used. All images were analyzed in gray scale. Before analysis, images for axonal transport analysis were randomized, so that the observer was masked to control and high-IOP eyes. Twelve nerve bundles in each eye were then followed from the prelaminar region to 3 mm behind the lamina cribrosa. In a sample window of constant size, the average pixel intensity per 1 μm² of RITC was calculated at specific points along each nerve bundle. Points sampled along the nerve bundle included the beginning of the prelaminar region (immediately after the inner limiting membrane), the midpoint of the lamina cribrosa, and the point immediately after the lamina cribrosa. The postlaminar region was sampled at 0.2-mm increments along the nerve bundle for the first millimeter and then at every millimeter for the next 2 millimeters. All points sampled along the nerve bundle were normalized and expressed as a percentage intensity of the point in the prelaminar region. Nerve bundles were divided into four nasal, four central, and four temporal nerve bundles during sampling, to allow analysis of regional differences in axonal transport. A direct comparison of the RITC intensity between the control and high-IOP eyes in different regions of the optic nerve was performed. 

Early studies of rapid axonal transport demonstrated that the quantities of tracer that reached points down the axon decrease in a logarithmic fashion, ²⁰ suggesting that a relatively constant proportion is retained or used at each point along the axon, with the remaining constant proportion transported distally. Subsequent modeling work which discusses various assumptions, based on experimental data also use exponential functions in describing tracer distribution along the nerve. ^{21 22} Hence, we assumed that under normal circumstances, a homogeneously transporting axon would maintain constant proportions of transport from one point to the next. We calculated these proportions to identify regions where axonal transport may differ significantly. 

As an estimate of relative axonal transport activity within different segments of the nerve the proportional change in RITC intensity per millimeter of nerve in each segment was determined. We calculated this by dividing the difference in RITC intensity between the sample point and that immediately preceding it by the distance separating these two points in millimeters. This result was expressed relative to the mean RITC intensity of these two points. A mathematical representation of this calculation is in this formula:  

\[\left(\frac{I_{\mathrm{B}}{-}I_{\mathrm{A}}}{D_{\mathrm{B}}{-}D_{\mathrm{A}}}\right)\left/\right.\left(\frac{I_{\mathrm{A}}{+}I_{\mathrm{B}}}{2}\right),\]

where I _B is the RITC intensity at the distal point; I _A is RITC intensity at the proximal point; D _B is the distance of the distal point from the inner limiting membrane (ILM; in millimeters); and D _A, is the distance of the proximal point from the ILM (in millimeters). 

This formula calculates the proportional change in RITC intensity per millimeter in that nerve segment. We used the result as an index of segmental axonal transport. This method allowed us to study the rate of change in RITC intensity from the prelaminar to the midpoint of the lamina cribrosa, from the midpoint of the lamina cribrosa to the immediate postlaminar region, and at fixed intervals along the postlaminar region. The changes in RITC intensity in nine separate segments in the control and high-IOP eyes were studied. Thus, in addition to comparing axonal transport rates between control and high-IOP eyes, we also were able to compare axonal transport rates within the different laminar regions and between central, nasal, and temporal nerve bundles of each eye. 

Reproducibility of Histologic Measurements

To determine observer reproducibility, 32 cytoskeletal images and two axonal transport images were quantified on three separate occasions, each at least 1 week apart, by the same masked observer who performed all the data analyses. The prelaminar, lamina cribrosa, and postlaminar regions of each cytoskeletal image were quantified. Six pairs of images for NFH and two pairs of images for each NFHp, NFM, NFL, tubulin, and MAP antibody were analyzed. Twelve nerve bundles from each axonal transport image were also quantified as described previously, with all points expressed as a percentage intensity of the prelaminar region. Each pair of images that were used for reproducibility analysis of cytoskeleton and axonal transport data consisted of the low- and high-IOP images from the same animal. A coefficient of variation (CV) was calculated for each cytoskeletal component and axonal transport data. Three-way analysis of variance (ANOVA) was used to test whether the difference in mean NFH intensity within each region for each measurement day was statistically significant. Three-way ANOVA was also used to test whether there was a difference in mean CV between the control and high-IOP eye within each region for each cytoskeletal antibody and axonal transport data. 

Statistical Analysis

All statistical testing was performed with commercial software (SigmaStat, ver. 3.1; SPSS, Chicago, IL). Both RITC and immunohistochemistry stain intensity data were found to be normally distributed, by Kolmogorov-Smirnov testing (P > 0.05). ANOVA was used to assess factor effects on the RITC labeling and cytoskeletal immunohistochemical staining, with post hoc factor comparison performed using a paired Student’s t-test with Bonferroni correction. 

Axonal Transport.

Three-way ANOVA was used to assess the effects of high or control IOP, individual pig, and position within the nerve on RITC intensity and segmental axonal transport in central, nasal, and temporal nerve bundles (nerve bundle location). Three-way ANOVA was used to assess the effects of nerve bundle location and individual pig on segmental axonal transport, at different positions along the nerve in each of the high-pressure and control IOP eyes. Three-way ANOVA was used to assess the effects of different nerve positions and individual pig on segmental axonal transport in the central, nasal, and temporal bundles in each of the high-pressure and control IOP eyes. 

Cytoskeleton.

Three-way ANOVA was used to assess the effect of high or control IOP, optic nerve region (prelaminar, lamina cribrosa, and postlaminar), and individual pig on each cytoskeletal antibody stain intensity. Because we had seven images aligned in superior-inferior order for each cytoskeletal antibody in each eye, we were interested in investigating whether there was a significant difference in the cytoskeletal intensity between different levels of the optic nerve along the z-plane within the same eye. To determine this, we used a three-way ANOVA to assess the effect of level of optic nerve slice, individual pig, and optic nerve region on each cytoskeletal antibody stain intensity in each of the high-IOP and control IOP eyes. 

Measurement Reproducibility.

Coefficient of variation results for observer reproducibility measurements were found to be non-normal, and so ANOVA on ranks was used to compare them and analyze the influence of various factors. The Tukey test was used for post hoc paired analysis. 

Results

General

The mean blood pressure in six pigs was 83.1 ± 5.9 mm Hg. Average arterial pO₂ was 100.2 ± 3.1 mm Hg, pCO₂ was 36.8 ± 0.8 mm Hg, and pH was 7.5 ± 0.0 on blood gas analysis. The mean CSFp was 5.5 ± 1.8 mm Hg. The average left- and right-eye IOP was 43.7 ± 1.1 and 13.2 ± 0.9 mm Hg, respectively. The average differences between the IOP and CSFp in the left and right eyes were 37.7 ± 2.2 and 7.1 ± 1.1 mm Hg, respectively. Mean experimental data for the individual pigs are presented in Table 1 . 

Reproducibility of Histologic Measurements

The mean CV for the cytoskeletal antibody stains was small, at 3.3% (SD 1.9%), with no significant difference between that of the three regions (two-way ANOVA on ranks; P > 0.05). Some difference in CV was found between the different cytoskeletal stains (two-way ANOVA on ranks; P < 0.001) with NFH (CV, 5.3%) being significantly different from tubulin (CV, 1.9%, P < 0.001), NFM (CV, 2.3%, P < 0.001), and NFL (CV, 2.5%, P < 0.001). Repeated measurements on six pairs of NFH data found no relationship between day of measurement and stain intensity in any of the three laminar regions (three-way ANOVA, P > 0.05). Testing the influence of antibody type and disc location and whether high-IOP or control revealed no significant difference in CV between high IOP and control eyes (three-way ANOVA, P = 0.207). 

Mean CV of the axonal transport RITC measurements was 4.4%, with significant variation along the nerve (one-way ANOVA on ranks, P < 0.001). There was greater variation in intensity in the region more than 2 mm posterior to the lamina cribrosa (CV, 12.1%) than in the lamina cribrosa (CV, 1.7%, P < 0.001) or the immediate postlaminar region (CV, 2.9%, P < 0.001), where the intensity tended to be greater. Using two-way ANOVA on ranks, we found that high IOP or control IOP had no influence on CV (P = 0.145). 

Optic Nerve Head Measurements

The mean prelaminar thickness from our experiments was 285.2 ± 23.7 μm. The mean nasal, central, and temporal lamina cribrosa thickness from our experiments were 358.4 ± 32.7, 380.7 ± 31.3, and 354.1 ± 33.7 μm, respectively, with the average midpoint of the lamina cribrosa being 475 μm from the ILM. Mean lamina cribrosa thickness measurements in the nasal, central, and temporal region in the control and high-IOP eye for each pig is presented in Table 2 . There was no significant difference in the prelaminar thickness between the control and high-IOP eyes (P = 0.4). The mean lamina cribrosa thickness in the high-IOP eyes was greater than that in the control eyes in four of six animals; however, the differences in mean did not reach formal statistical significance (two-way ANOVA, P = 0.51). It should be noted that our study was not designed to clarify any possible change in lamina thickness. 

Axonal Transport

Figure 3is a representative image taken 6 hours after RITC injection into the vitreous cavity of both control and high-IOP eyes. The intensity of RITC was visibly greatest in the prelaminar region of both control and high-IOP eyes. The distribution of RITC in the control eye appeared more diffuse and homogeneous when compared to the high-IOP eye. Furthermore, RITC traveled farther into the postlaminar region in the control eye with the intensity decreasing as the distance from the lamina cribrosa increased. There was visibly less RITC staining in the postlaminar tissue in the high-IOP eye when compared to the control eye (Fig. 3A) . 

Axonal Transport within the Control and High-IOP Eyes.

When segmental axonal transport at different points along the optic nerve between the nasal, temporal, and central nerve bundles was compared in the control eye, the rate of axonal transport was found to be significantly different in the lamina cribrosa region (three-way ANOVA, P < 0.001). There was a significant difference in RITC intensity between the central, nasal, and temporal regions within the lamina cribrosa region. Post hoc testing revealed that the difference existed only between central and nasal (P < 0.01), and central and temporal (P < 0.001), but not temporal and nasal bundles (P > 0.11) in this region. Post hoc testing revealed a significant decrease in segmental axonal transport within the lamina cribrosa portion of the nasal and temporal nerve bundles of the control eye (P < 0.001). Within the central nerve bundles of the normal-pressure eye there were no significant differences in segmental axonal transport between the prelaminar, lamina cribrosa, and any of the postlaminar regions (three-way ANOVA, P > 0.05; Fig. 4B ). When the same analysis was performed on the nasal and temporal nerve bundles of the normal-pressure eyes the segmental axonal transport in the prelaminar, lamina cribrosa, and immediate postlaminar regions were significantly different from each other (three-way ANOVA, P < 0.001; Fig. 4B ). Post hoc testing revealed a decrease in segmental axonal transport within the lamina cribrosa portion of the nasal and temporal nerve bundles of the normal-pressure eye (P < 0.001). 

When segmental axonal transport at different points along the optic nerve in the nasal, temporal, and central nerve bundles were compared in the high-IOP eyes, the rate of axonal transport was found to be significantly different in the prelaminar, lamina cribrosa, and the anterior two postlaminar regions (three-way ANOVA, P < 0.001). There was a significant difference in RITC intensity in the nasal, central, and temporal locations within these regions. Post hoc testing revealed that the difference existed only between central and nasal (P < 0.001) and central and temporal (P < 0.001), but not temporal and nasal bundles (P > 0.08). Post hoc testing revealed a significant decrease in segmental axonal transport in these regions in the nasal and temporal nerve bundles of the high-IOP eye (P < 0.001). Segmental axonal transport between points in the prelaminar, lamina cribrosa, and postlaminar regions of the central nerve bundles of the high-IOP eyes was found to vary significantly (three-way ANOVA, P < 0.001; Fig. 5B ). Post hoc testing revealed significant reduction in segmental axonal transport in the lamina cribrosa segment and also the 0.2- and 0.4-mm postlaminar segments (all P < 0.001). Segmental axonal transport between points in the prelaminar, lamina cribrosa, and postlaminar regions of the nasal and temporal nerve bundles of the high-IOP eyes was also found to vary significantly (three-way ANOVA, P < 0.001; Fig 4B ). Post hoc testing revealed a significant reduction in segmental axonal transport in the lamina cribrosa and the anterior two regions in the postlaminar nerve (all P < 0.001). In the nasal, temporal, and central nerve bundles of the high-IOP eye, axonal transport was most reduced at the lamina cribrosa portion of the nerve (Figs. 4B 5B) . 

Examining results from individual pigs revealed that five of six animals had reduced segmental axonal transport rates in the nasal and temporal nerve bundles in comparison to central nerve bundles within the lamina cribrosa in the control eye, with three of these differences reaching formal statistical significance (P < 0.019). Again, five of six animals had reduced axonal transport rates in the nasal and temporal nerve bundles in comparison to central nerve bundles in the prelaminar region, lamina cribrosa, and anterior 0.4-mm postlaminar nerve in the high-IOP eye, with three of these differences reaching formal statistical significance (all P < 0.024). Within the lamina cribrosa all six animals had reduced segmental axonal transport rates in comparison to the prelaminar and immediate postlaminar regions in both the control and high-IOP eyes, with five of these differences reaching formal statistical significance (all P < 0.003). In the high-IOP eyes of five animals, the rate of segmental axonal transport in the 0.2- and 0.4-mm postlaminar segments was significantly reduced compared with the prelaminar rate (all P < 0.011). 

Axonal Transport Comparisons between Control and High-IOP Eyes.

When the intensity of RITC in control and high-IOP eyes at points along the central nerve bundles were compared by using ANOVA with post hoc analysis, there was a statistically significant decrease in intensity in the high-IOP eye at all points between the lamina cribrosa and 2 mm of postlaminar tissue (three-way ANOVA, P < 0.001; Fig. 5A ). When a similar comparison with post hoc analysis was performed on the nasal and temporal nerve bundles the intensity of RITC was significantly lower in the lamina cribrosa and all points up to 3 mm of postlaminar tissue in the high-IOP eye (three-way ANOVA, P < 0.001; Fig. 4A ). When graphically representing data in Figures 4A and 5A , the prelaminar point was denoted as 0 μm from the ILM and the midpoint of the lamina cribrosa being 475 μm from the ILM. Because there was no significant difference between the nasal and temporal data, we combined those data and termed them peripheral in Figure 4 . 

Figure 5Bshows the segmental axonal transport rate along the central nerve bundles in different regions of the optic nerve in both control and high-IOP eyes. Comparisons using ANOVA with post hoc analysis showed that a statistically significant difference in central bundle axonal transport rate only occurred in the lamina cribrosa region (three-way ANOVA, P < 0.001, Fig. 5B ). The rate of axonal transport was lower in the high-IOP eye. Figure 4Bshows the axonal transport rate along the nasal and temporal nerve bundles in different regions of the control and high-IOP eyes. ANOVA testing with post hoc analysis revealed that nasal and temporal bundle axonal transport rates were significantly decreased in the high-IOP eye at points 0.238 mm from the ILM, 0.570 mm from the ILM (lamina cribrosa region), and 0.765 and 0.965 mm from the ILM (first 0.4 mm of postlaminar tissue), as shown in Figure 4B(three-way ANOVA, P < 0.001). 

Examining results from individual pigs revealed that all six had significantly (all P < 0.01) reduced segmental axonal transport rates in the high-IOP eye compared with the control eye within the anterior laminar region. Within the posterior laminar region all six had reduced segmental axonal transport rates in the high-IOP eye compared with the control eye with differences in four reaching formal statistical significance (all P < 0.025). Within the anterior 0.2-mm postlaminar region, again all six pigs had reduced segmental axonal transport rates in the high-IOP eye compared with the control eye, with two differences reaching significance (all P < 0.001). Within the anterior 0.2 to 0.4 mm postlaminar region, again all six had reduced segmental axonal transport rates in the high-IOP eye compared to the control eye with three differences reaching significance (all P < 0.028). 

Cytoskeleton

Results of three-way ANOVA examining the effect of level of optic nerve slice, individual pig, and optic nerve region on cytoskeletal antibody stain intensity found no significant difference between the vertical levels with the use of any of the cytoskeletal antibodies, in both the control and high-IOP eyes (P > 0.05). 

NFHp Staining.

Figure 6Ashows NFHp staining in the porcine optic nerve. In the high-IOP eye there was less stain visible in all three laminar regions than in the control eye. Three-way ANOVA with post hoc testing demonstrated a statistically significant decrease in intensity of NFHp stain across the entire optic nerve between the high-IOP group in comparison with the control when allowing for individual pig variation and optic nerve region variation (P < 0.001; Fig. 7 ). Subanalysis revealed significantly less stain in the prelaminar (P < 0.001), lamina cribrosa (P < 0.001), and postlaminar (P < 0.001) regions in the high-IOP eyes than in normal-IOP eyes. Examining results from the individual pigs revealed that all six had significantly (all P < 0.004) reduced stain intensity in the high-IOP eye compared with the control eye within the lamina cribrosa and postlaminar regions. In the prelaminar region, five of six animals demonstrated this difference (all P < 0.001). 

NFH Staining.

Figure 6Bshows NFH staining in the porcine optic nerve where visibly less stain was seen in the high-IOP eye than in the control eye. Three-way ANOVA with post hoc testing demonstrated a statistically significant difference in NFH stain intensity across the entire optic nerve between the high-IOP group in comparison to the control, when adjustment was made for individual pig variation and optic nerve region variation (P < 0.001, Fig. 7 ). Subanalysis revealed significantly less stain in the prelaminar (P < 0.001), lamina cribrosa (P < 0.001), and postlaminar (P < 0.001) regions in the high-IOP eyes than in normal IOP eyes. Examining results from individual pigs revealed that all six had significantly (all P < 0.024) reduced stain intensity in the high-IOP eye compared with the control eye within the prelaminar region. In the lamina cribrosa and postlaminar regions five of six animals demonstrated this difference (all P < 0.001). 

NFM Staining.

Figure 6Cshows NFM staining in the porcine optic nerve. In the high-IOP eye there was visibly less NFM staining in the prelaminar, lamina cribrosa, and postlaminar regions than in control eyes. Three-way ANOVA with post hoc testing demonstrated a statistically significant difference in NFH stain intensity across the entire optic nerve between the high-IOP group in comparison to the control group, when adjustment was made for individual pig variation and optic nerve region variation (P < 0.001, Fig. 7 ). Subanalysis revealed significantly less stain in the prelaminar (P < 0.001), lamina cribrosa (P < 0.001), and postlaminar (P < 0.001) regions in the high-IOP eyes than in normal-IOP eyes. Examining results from individual pigs revealed that all six had significantly (all P < 0.013) reduced stain intensity in the high-IOP eye than in the control eye within the prelaminar region. In the lamina cribrosa and postlaminar regions, five of six animals demonstrated this difference (all P < 0.027). 

NFL Staining.

Figure 6Dshows NFL staining in the porcine optic nerve. In the control eye, the intensity of NFL staining appeared visibly similar in the prelaminar, the lamina cribrosa, and postlaminar regions in both the control and the high-IOP eyes. Three-way ANOVA with post hoc testing showed no significant difference in NFL stain intensity in any of the three regions of the optic nerve head in the high-IOP eye in comparison with the control (three-way ANOVA; P > 0.05; Fig. 7 ). 

Tubulin Staining.

The pattern of tubulin staining was visibly similar in both the high-IOP and the control eyes (Fig. 6E) . Three-way ANOVA with post hoc testing showed no significant difference in tubulin stain intensity in any of the three regions of the optic nerve head in the high-IOP eye in comparison with the control (three-way ANOVA, P > 0.05; Fig. 7 ). 

MAP Staining.

The distribution of MAPs was visibly similar in the control and high-IOP eyes (Fig. 6F) . Three-way ANOVA with post hoc testing showed no significant difference in MAP stain intensity in any of the three regions of the optic nerve head in the high-IOP eye in comparison with the control (three-way ANOVA, P > 0.05; Fig. 7 ). 

Discussion

The purpose of this study was to determine whether elevated IOP induces corresponding changes in axonal transport and the axonal cytoskeleton in the prelaminar, lamina cribrosa, and postlaminar regions of the optic nerve. CSFp and IOP are the two major determinants of the translaminar pressure gradient. ²³ We used RITC as a tracer to study axonal transport in a porcine model of high IOP where the IOP and CSFp were continually monitored. The major findings from this study are as follows: (1) The lamina cribrosa is a point of axonal transport decrease in the control and high-IOP eyes; (2) high IOP caused a significant reduction in proportional change of segmental RITC intensity in the prelaminar, lamina cribrosa, and postlaminar regions of the optic nerve head, suggesting that axonal transport in these segments was altered; (3) high IOP produced notable changes in the axonal cytoskeleton. There was selective impairment of NFHp, NFH, and NFM in the prelaminar, lamina cribrosa, and postlaminar regions after 6 hours of high IOP. 

Glaucoma is a slowly progressive optic neuropathy that is characterized by the excavated appearance of the optic nerve head, associated with the loss of RGCs. It is a disease defined by specific morphologic changes in the retina and optic nerve head. Increased IOP is a major risk factor predictive of visual loss due to RGC damage in glaucoma. ¹ Our model of high-IOP-induced axonal alteration allows the investigation of the dynamics of initial morphologic, molecular, and functional changes under controlled conditions. However, this study did not explore the long-term pathologic processes that occur in human glaucoma. Our conclusions should therefore be interpreted only as giving insight into the effects of high IOP on axonal transport and cytoskeleton and the possible underlying mechanisms of axonal damage during the early stages of glaucoma. 

In previous studies of axonal transport changes in the optic nerve, tracers have been used that necessitate extensive tissue processing for analysis. ^{13 14 17 24} The processing often results in the loss of spatial detail within the nerve, rendering simultaneous labeling of other structures within the same tissue impossible. Consequently, it has been difficult to correlate changes in axonal transport with morphologic changes that occur in the optic nerve. RITC has been used to study the neural connections of the visual system in different animal species by injection into the vitreous. ^{18 25 26} It is a fluorescent tracer that is incorporated into proteins and is transported in both anterograde and retrograde directions by axonal transport. ²⁷ It has the advantage of being visualized by fluorescent methods and permits labeling of other structures within the same tissue by using immunohistochemical techniques. For these reasons we used RITC to study axonal transport. We also found quantitation of RITC intensity along the optic nerve to have a high degree of reproducibility. 

An IOP increased to 43 mm Hg for 6 hours in the porcine eye caused a significant reduction in axonal transport in the optic nerve which is consistent with previous primate work. ¹⁷ The greatest change in RITC intensity was seen across the lamina cribrosa in both control and high-IOP eyes. These data support previous work and suggest that the lamina cribrosa acts as a choke point for axonal transport, even in the normotensive eye, with this effect being exaggerated when the IOP is increased. ^{14 28} In line with previous reports, we also observed regional differences in axonal transport between the central, nasal, and temporal nerve bundles after an increase in IOP. Axonal transport blockade was previously reported to be most pronounced in the nasal and temporal quadrants of the optic nerve head during acute ocular hypertension in the monkey. ¹⁴ Similarly, in a model of ocular hypertension in the albino rabbit, the blockage in axonal transport was reported as being most marked in the marginal regions of the optic disc. ²⁴ The porcine optic disc is elliptically shaped with the long axis along the horizontal meridian, without any significant variation in laminar pore size, whereas the human optic disc is also elliptically shaped but with the long axis generally along the vertical meridian (Fig. 1)and with larger pores within the superior and inferior temporal regions. ²⁹ We examined axonal transport differences between the nasal, central, and temporal regions of the optic disc in sections cut along the horizontal axis. Using the horizontal axis allowed us to maximize the spatial separation between the central, nasal, and temporal nerve bundles. Differences in the axonal transport between superior and inferior regions were not assessed. It should also be noted that we did not study bilaterally normal porcine eyes, and so we do not know how much physiologic difference between the two eyes of such animals might be present. Thus, some portion of these differences may not be due to high IOP, but rather to physiologic differences between two normal eyes of the same animal and may be unaccounted for in the post hoc paired testing. 

We observed a decrease in axonal transport in the lamina cribrosa region in both the central and nasal and temporal nerve bundles of the high-IOP eye in comparison to control eyes. However, in addition to the lamina cribrosa, we also observed axonal transport to be significantly different in the prelaminar and first 0.4 mm of postlaminar tissue in the nasal and temporal nerve bundles of the high-IOP eyes in comparison to the control eyes, which implies that an increase in IOP had a more pronounced affect on axonal transport in the nasal and temporal nerve bundles. Axonal transport within the normotensive eye was also decreased in the lamina cribrosa region of the nasal and temporal nerve bundles, with no significant reduction observed within the central nerve bundles. It is difficult to satisfactorily explain the different segmental axonal transport rates between the nasal, temporal, central bundles of the high- and normal-IOP eyes. Models in which potential neural strain is calculated within the prelaminar, lamina cribrosa, and postlaminar regions suggest an uneven distribution with a possible increase in the nasal and temporal zones of these three regions. ³⁰ Although we do not have a definite explanation for the difference between central and nasal and temporal nerve bundles, it is unlikely that acute scleral canal deformation is responsible for the changes observed as the scleral canal is not known to expand in monkeys with acute IOP elevation. ³¹ Previous work has demonstrated that the pressure gradient between the intraocular and cerebrospinal fluid compartments extends across the lamina cribrosa, without evidence of extension into the postlaminar region. ³² We cannot provide a confident explanation of why inhibition of axonal transport extends into the postlaminar nerve in the nasal and temporal nerve bundles. However, it is likely that there are greater metabolic demands within the lamina cribrosa, ³³ leading to the possibility of this demand’s affecting adjacent regions when the IOP is elevated. 

There was a dramatic change in the neurofilament subunits after 6 hours of raised IOP. We observed a significant decrease in NFH, NFHp, and NFM across all three regions of the optic nerve head in the high-pressure eye. It must be noted that our confocal imaging of the cytoskeleton extended only 0.2 mm posterior to the lamina cribrosa, and so we cannot comment on whether cytoskeletal changes occurred farther posteriorly. Each neurofilament subunit has a distinct function. Selective deletion or overexpression of neurofilament subunits can result in variable forms of disease that affect axonal structure and function. ¹¹ Changes in NFHp and NFL have already been described in separate studies of the optic nerve using primate glaucoma models. ^{15 34} To our knowledge there are no reports that examine parallel changes in all neurofilament subunits after an acute increase in IOP. 

Neurofilaments are extensively cross-linked with each other and with other cytoplasmic components through sidearms. Dephosphorylation of these sidearms alters the charge and the mechanical properties of these crossbridges, making them more pliant and more likely to unfold. ³⁵ This effect could result in an axonal cytoskeletal structure in which the neurofilaments are oriented randomly, less well connected, and sometimes fascicled. ³⁶ Consequently, there may be disruption of the cytoskeletal network along which molecular motors travel manifesting as abnormalities in axonal transport. Phosphorylation of neurofilaments has also been reported to protect them from proteolysis. ³⁷ It is possible that the initial insult to the cytoskeleton is a change in the phosphorylation status of NFM and NFH. As these proteins become gradually dephosphorylated, the cytoskeleton may become increasingly vulnerable to proteolysis as observed by a reduction in total NFH. NFL forms the backbone of the neurofilament heteropolymer and hence may be initially shielded from proteolytic activity by the NFM and NFH associated with it, which may explain the absence of NFL changes observed in this study. 

A reduction in microtubule number and MAPs in the region of the lamina cribrosa has been reported after 4 hours of raised IOP. ³⁸ This work was performed on guinea pigs that have an avascular retina as well as a less well-structured lamina cribrosa in comparison to that of the pig. ³⁹ The experimental eye in this work was also elevated to an IOP of 60 mm Hg. The differences in animal species and experimental design may account for the difference in results seen between our work and that previously published. Previous work by Fernandez et al. ⁴⁰ has demonstrated a reduction in axonal transport despite the presence of intact microtubules in the crayfish nerve cord. Our results were consistent with this latter finding. We found no change in the intensity of MAP or tubulin after 6 hours of elevated IOP, despite a reduction in axonal transport. 

This work demonstrates that axonal transport and cytoskeletal changes follow a similar distribution in the optic nerve head after 6 hours of raised IOP. The optic disc deforms posteriorly in ocular hypertension. ⁴¹ This deformation, in combination with an increased pressure gradient may create sufficient shear and other forces on the cytoskeleton to result in some of the changes seen in this study. An alteration in energy supply and demands to this region as a result of increased IOP may also cause and exacerbate this problem. As the cytoskeleton is involved in the pathway along which axonal transport takes place, it is possible that a change to its structure and composition contributes to the axonal transport changes that occur in glaucoma. These studies of acute IOP-induced axonal transport abnormalities lay the foundation for understanding the more long-term processes present in human glaucoma. 

 Supported by the National Health and Medical Research Council of Australia and the Australian Research Council Centre of Excellence in Vision Science.

 Submitted for publication August 23, 2006; revised February 6 and March 22, 2007; accepted June 4, 2007.

 Disclosure: C. Balaratnasingam, None; W.H. Morgan, None; L. Bass, None; G. Matich, None; S.J. Cringle, None; D.-Y. Yu, 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.

 

Clinical fundus photographs of a porcine and human optic nerve head. Left: an image of a porcine optic nerve with fenestrated lines demonstrating the region of tissue sectioned for cytoskeleton and axonal transport studies. Right: a healthy optic disc from a 28-year-old man provided for comparison.

Figure 2.

 

Van Gieson stain of a longitudinal section of porcine optic nerve, illustrating the prelaminar (PL), lamina cribrosa (LC), and postlaminar (PoL) regions of the nerve. Dotted lines: the anterior and posterior boundaries of the lamina cribrosa used to calculate optic nerve head measurements. Ch, choroid; ILM, inner limiting membrane; NFL, nerve fiber layer; Sc, sclera. Scale bar, 500 μm.

Table 1.

 

Physiological Data for Individual Pigs

	BP	(L) IOP	(R) IOP	CSFp	pO2	pCO2	pH
Pig 1	87.6 ± 2.2	45.0 ± 0.6	11.4 ± 0.4	3.5 ± 0.2	91.4 ± 5.5	33.3 ± 1.4	7.5 ± 0.0
Pig 2	81.1 ± 1.9	43.3 ± 0.7	11.6 ± 0.5	6.1 ± 0.7	93.6 ± 6.6	35.8 ± 1.8	7.5 ± 0.0
Pig 3	83.6 ± 1.4	41.5 ± 0.5	12.8 ± 0.4	6.7 ± 0.6	93.8 ± 6.3	38.3 ± 0.6	7.5 ± 0.0
Pig 4	86.0 ± 2.0	43.9 ± 0.5	14.2 ± 1.2	6.8 ± 0.4	111.2 ± 5.2	34.0 ± 0.6	7.5 ± 0.0
Pig 5	72.9 ± 1.5	43.4 ± 0.5	14.9 ± 0.5	5.9 ± 0.9	105.3 ± 11.6	40.8 ± 2.3	7.4 ± 0.0
Pig 6	90.9 ± 1.5	45.0 ± 0.8	14.2 ± 1.3	5.3 ± 0.5	110.0 ± 4.5	38.5 ± 1.4	7.5 ± 0.0
Mean	83.1 ± 5.9	43.7 ± 1.1	13.2 ± 0.9	5.5 ± 1.8	100.2 ± 3.0	36.8 ± 0.8	7.5 ± 0.0

 The mean (± SE) for each physiological variable is given also. All measurements apart from pH are in mm Hg.

Table 2.

 

Laminar Thickness Measurements for the Individual Pigs

	Nasal			Central			Temporal
		Control IOP	High IOP	Control IOP	High IOP	Control IOP	High IOP
Pig 1	328.6	333.5	340.1	368.7	331.2	329.4
Pig 2	331.3	323.1	344.4	339.5	326.6	308.4
Pig 3	348.2	356.1	396.3	382.4	353.1	348.7
Pig 4	368.1	390.4	388.7	411.3	374.3	392.6
Pig 5	338.2	351.7	356.8	394.4	339.7	330
Pig 6	411.7	420	424.3	453.2	403.7	411.9
Mean	354.3 ± 12.9	362.5 ± 14.9	375.1 ± 13.6	391.6 ± 15.8	354.8 ± 12.0	353.5 ± 16.5

 Thickness data are expressed in micrometers. The mean ± SEM is shown.

Figure 3.

 

Axonal transport in the control and high-IOP eyes. (A) Confocal images of RITC transport after 6 hours of raised IOP. Left: control eye; right: high-IOP eye. RITC traveled well past the lamina cribrosa in the control eye, whereas there was accumulation of tracer in the prelaminar and lamina cribrosa regions of the high-IOP eye, with little transport past the lamina cribrosa. (B) Van Gieson-stained sections taken from similar regions of the RITC-labeled optic nerve are provided for reference. Left: control eye; right: high-IOP eye. Dotted lines: lamina cribrosa region in each image. Scale bar, 400 μm.

Figure 4.

 

Relationship between averaged RITC intensity and distance from prelaminar region along the optic nerve in the peripheral nerve bundles (nasal and temporal data combined) of the control and high-IOP eyes. (A) RITC intensity was normalized and expressed as a percentage intensity of the prelaminar value. There was a significant reduction in RITC intensity at all points from the lamina cribrosa up to 3 mm of postlaminar tissue in the high-IOP eye when compared with the control eye. (B) Proportional change in RITC intensity within different segments of the peripheral optic nerve. Segmental axonal transport was significantly decreased in the prelaminar region, midpoint of the lamina cribrosa, and two anterior postlaminar points in the high-IOP eye in comparison to the control eye. *Significant difference with P < 0.001 as determined by three-way ANOVA with post hoc testing.

Figure 5.

 

Relationship between averaged RITC intensity and distance from the prelaminar region along the optic nerve in the central nerve bundles of the control and high-IOP eyes. (A) RITC intensity is normalized and expressed as a percentage intensity of the prelaminar value. There was a significant reduction in RITC intensity at all points between the lamina cribrosa and 2 mm of postlaminar tissue in the high-IOP eye when compared with the control eye. (B) Proportional change in RITC intensity within different segments of the central nerve—an index of regional axonal transport. Segmental axonal transport was significantly decreased at the midpoint of the lamina cribrosa in the high-IOP eye in comparison to the control eye. *Significant difference with P < 0.001 as determined by three-way ANOVA with post hoc testing.

Figure 6.

 

Confocal images of neurofilament stains. Left: control eye; right: high-IOP eye. The prelaminar, laminar, and postlaminar regions of the nerve are labeled. (A) NFHp, (B) NFH, (C) NFM, (D) NFL, (E) tubulin, and (F) MAP stains. Scale bar, 100 μm.

Figure 7.

 

Comparison of average intensities of cytoskeletal proteins in each region of the optic nerve. (A) Prelaminar region, (B) lamina cribrosa region, and the (C) postlaminar region. There is a significant reduction in NFH, NFHp, and NFM across all three regions in the high-IOP eye. There was no difference in NFL, tubulin, and MAP in any of the regions (n = 42). **Significant difference with P < 0.001 as determined by three-way ANOVA with post hoc testing.

The authors thank Peter Gray, Department of Veterinary Anesthetics, Veterinary Hospital, Murdoch University, for assistance with the anesthetic protocol; Dean Darcey and Judi Granger for technical assistance; Hsien Chan for assistance with statistical analysis; and Paul Rigby from the Biomedical Imaging and Analysis Facility, University of Western Australia, for assistance with microscopy.