| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1From the Departments of Ophthalmology, 2Anatomy and Neurobiology, and 3Genetics, University of Maryland School of Medicine, Baltimore, Maryland.
 |
Abstract |
|---|
 Top Abstract MethodsResultsDiscussionReferences |
|---|
METHODS. A novel rodent photoembolic stroke model was developed that is directly analogous to human AION. Using histologic, electrophysiological, molecular- and cell biological methods, the early changes associated with isolated RGC axonal ischemia were characterized.
RESULTS. Functional (electrophysiological) changes occurred in RGCs within 1 day after rAION, with a loss of visual evoked potential (VEP) amplitude that persisted in the long term. The retinal gene expression pattern rapidly changed after rAION induction, with an early (<1 day) initial induction of c-Fos mRNA, and loss of RGC-specific gene expression. RGC-specific protein expression declined 2 days after detectable mRNA level changes, and immunostaining suggested that multiple retinal layers react to isolated RGC axonal ischemia.
CONCLUSIONS. rAION rapidly results in electrophysiological and histologic changes similar to clinical AION, with reactive responses in primary and supporting neuronal cell layers. The rAION model can enable a detailed analysis of the individual retinal and optic nerve changes that occur after optic nerve stroke, which may be useful in determining possible therapeutic interventions for this disorder.
Numerous models have been created to identify the RGC response to ON damage, including ON crush,4 transection,5 increased ocular pressure,6 and infusion of vascular constricting agents.7 However, none of these models resembles the natural course of AION, or CNS ischemic axonopathy in general. We therefore developed an easily utilizable, photothrombosis-induced rat model of AION (rodent AION, or rAION), which resembles the functional, histologic, and physiologic defects seen in the human disease.
We selectively thrombosed the microvessels supplying the ON, by directly illuminating the intraretinal portion of the ON with a laser, after intravenous infusion with the photosensitizing agent rose bengal (RB), a derivative of fluorescein dye. RB is activated by visible light to generate superoxide radicals.8 Photoactivation of intravascular RB selectively damages the vascular endothelium, producing thrombosis while sparing nonvascular tissues.9 10 11 12 The RB-photoactivation technique has been demonstrated to cause retinal vascular thrombosis only in areas of direct light exposure.12 A study has also demonstrated that it is superoxide radicals, rather than direct thermal damage, that are the primary cause of light-induced, RB cellular toxicity.13 Although superoxide radicals are toxic to all vascular membranes, appropriate RB exposure times can thrombose capillaries supplying just the ON, while sparing the larger caliber central retinal vessels that pass through the ON and supply the inner retinal circulation. The rAION model is thus directly analogous to human AION. We used this method to determine the in vivo RGC and ON response to rAION, without significant intraretinal damage or disturbance of underlying retinal structures.
|
Methods |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
Histologic Preparation
Retinal tissue for histologic analysis was fixed in either 4% paraformaldehyde-phosphate-buffered saline (PF-PBS) or methacarn and paraffin embedded. Six-micrometer sections were step-cut through rAION-treated and control eyes, and cells in the RGC layer, as well as the number of cell layers in the inner (INL) and outer (ONL) nuclear retinal layers, were counted (12 high-power fields per retina). Statistical analysis was performed (n = 5 animals/group) comparing total number of RGCs per section and the relative thickness in cell layers of the INL and ONL, in each test group for both treated and contralateral eyes, using Student’s two-tailed t-test.
Visualization of Optic Nerve Circulation
To confirm the ischemic nature of the RB laser-induced ON lesion, control animals, and animals 30 minutes after rAION induction were terminally anesthetized with 50 mg/kg pentobarbital (Nembutal; Abbott Laboratories, Abbott Park, IL). The inferior vena cava was incised, and the left ventricle was rapidly perfused with 5 mL India ink through a 19-gauge intracardiac needle. The mouse was decapitated 10 seconds after beginning perfusion. This technique results in filling of all capillary beds (retinal, choroidal, and iris) with India ink before decapitation, allowing tracing of patent vessels. After death, eyes were dissected, and postfixed in 4% PF-PBS for 24 hours. Retinas were dissected, along with the choroidal vasculature immediately adjacent to the ON, flatmounted with aqueous mounting medium (Crystal/Mount; Biomeda Corp., Foster City, CA) and photographed.
Immunohistology and Optic Nerve Analysis
Optic nerve tissue was fixed in glutaraldehyde-paraformaldehyde fixative and embedded in Epon. Sections (0.5-µm-thick) of control and rAION-treated nerves were stained with toluidine blue. Immunohistology was performed with rabbit polyclonal antibodies specific for HSP84 and -86 proteins (ABR, Golden, CO) at a 1:100 dilution. Brn 3.2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:25 dilution. Sections were developed with a kit (Vectastain elite; Vector Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole (AEC) reagent.
Physiological Testing
Optic nerve function was evaluated on ketamine and /xylazine (80 mg and 4 mg/kg, respectively) anesthetized animals, through visual evoked potential (VEP). The fellow eye was covered with an opaque contact lens during testing. The stimulus was a 2 Hz, 1.95-cd s/m2 Ganzfeld strobe flash; 80 responses per eye were averaged. Band-pass cutoff frequencies were 0.3 and 100 Hz. The occipital (visual) cortical response was measured using electrodes placed on the shaved skull. The untreated (control) eye of each animal was used for internal comparison.
RNA Isolation and Gene Expression Analysis
Animals for RNA analysis were killed with CO2 inhalation. Retinal tissue was immediately dissected and stored at -70°C until use. RNA was extracted (RNAzol B; Tel-Test, Friendswood, TX). First-strand cDNA was generated from total RNA using a kit (Retroscript; catalog no. 1710; Ambion, Austin, TX). We characterized changes in mRNA levels of six genes, using a two-step, multiplex, semiquantitative reverse-transcriptase-based polymerase chain reaction (rt-PCR). Quantification of selected genes was compared using simultaneous amplification of the test gene, and an 18s ribosomal RNA loading standard (18s rRNA) cDNA product to compensate for tube-to-tube variations in RNA loading and PCR reactions. Because cellular 18s rRNA concentration is at least two orders of magnitude greater than individual messengers, 18s rRNA-cDNA product for each reaction was quantitatively inhibited by a selective competitive inhibitor of the 18s rRNA product (Competimer Technology; Ambion). The ratios of 18s rRNA primer and competimer were optimized for each test gene primer pair (Table 1) , to ensure that both the gene of interest and 18s rRNA product are measurable within the linear quantitation range. Gene expression profiles were compared between individual control (RB/no rAION induction) and rAION-induced retinal pairs. Gene-specific oligonucleotide primers were generated for individual rat sequences. These are seen in Table 1 . The studied genes c-fos, brn3.2, HSP70-1/2, opsin, HSP86, and HSP84 were chosen because: (1) c-fos is an indicator of immediate early tissue stress14 15 ; (2) brn3.2 is expressed specifically in the retina by RGCs16 ; (3) HSP70-1/2 is a tissue stress-induction marker used in many systems17 ; (4) HSP86 and -84 play major roles in RGC axonal function18 ; and (5) rod opsin is an indicator of outer retinal cell layer function and photoreceptor damage.19
|
|
Results |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
|
Effect of RB-Laser Induction Technique on Capillaries Supplying the ON
The rat ON is perfused by capillaries derived from the central retinal vessels, as well as recurrent choroidal vessels surrounding the nerve.21 Optic nerve capillary filling by India ink is seen in Figure 2 . In the sham-treated (laser/no dye) eye, many intraretinal capillaries supplied by the central retinal vessels were visible (Fig. 2A , small arrow). Within the ON (Fig. 2B) , there were many small capillaries. These are visible within the circle of the choroidal vessels that define the ON borders (Fig. 2B , large arrow). No difference was seen in filling characteristics of naïve control or RB-treated/no laser animals compared with sham-treated animals (data not shown). The intraretinal capillaries of rAION-induced animals (30 minutes after induction) away from the ON also fill with India ink (Fig. 2C , arrow), although there was incomplete filling of vessels close to the central vessels of the ON (Fig. 2C) . Examination of circulation within the ON of rAION animals showed a severe reduction in the filling of many of the ON capillaries (Fig. 2D) . Choriocapillary filling was clearly demarcated (Fig. 2D , double arrows), with few recurrent vessels visible between the central retinal vessels and choriocapillaris (Fig. 2D) . Thus, the ON defect induced by the RB-laser technique is a true ischemic lesion.
|
|
The VEP amplitude depression at early times after rAION induction (e.g., Fig. 3C , rat C; 3 days) continued even 45 days after induction (e.g., Fig. 3D , rat C; 45 days; dotted trace). No statistical analysis was performed at the latter time point, due to the low number of subjects. Thus, rAION produces a loss of ON electrical function, with permanent degradation of normal ON electrical activity.
rAION-Induced Histologic Changes in the Retina and ON
RGCs are present as a single cell layer in the rodent retinal (Fig. 4D ; RGC), and their axons compose the NFL (Fig. 4D) . NFL axons form the ON (Fig. 4A ; ON). The intraretinal portion of the ON was triangular in appearance and flat against the retinal surface (Fig. 4A , arrow), with little extrusion into the intraretinal compartment. One day after treatment, no changes in ON-retinal junction histology were seen in animals exposed to either laser illumination alone (Fig. 4A) or RB dye alone (data not shown). The NFL was triangular in appearance, with no displacement of the peripapillary retina (Fig. 4A , arrow). In contrast, NFL swelling anterior to the site of ON ischemia resulted in ON edema, 1 day after rAION induction, (Fig. 4B , double asterisks). Optic nerve edema was also apparent from the displacement of the peripapillary retina (Fig. 4B , double arrows). Thirty-seven days after induction, ON edema had resolved (Fig. 4C) . There was loss of the RGC axonal component of the intraretinal ON (Fig. 4C , arrow) with shrinkage of the apparent ON diameter. The intraorbital portion of the ON also showed increased cellularity centrally, suggesting ON remodeling (Fig. 4C , double arrows).
|
Quantification of average RGC nuclei in rAION-affected and different control retinas (n = 5 animals) is shown in Figure 5 . There was no difference in the average number of nuclei in the INL and ONL between any of the treated or naïve groups (measured as the total nuclear thickness, rather than individual nuclei (11.3 ± 0.7 layers for ONL; 3.9 ± 0.3 layers for INL).
|
Axonal Changes in ON Sections after rAION Induction
The normal (control) ON (Fig. 6A) contained regular axon bundles, with tightly packed myelinated large and small-caliber axons (Fig. 6A , Ax). At 6 days, There was also swelling and increased staining of individual axons (Fig. 6B , double arrows), suggesting either axonal swelling or edema. Eleven days after induction, there was axonal collapse, with disruption of normal architecture (Fig. 6C , arrow). Axon loss and stromal scarring were apparent 90 days after induction of rAION (Fig. 6D , asterisk). Post-rAION axonal loss in the ON was typically central, with sparing of the peripheral RGC axons (Fig. 6E) . Thus, both early and long-term functional and histologic changes occurred after induction of rAION that are similar to those in the human disease.3 24
|
50% of control (contralateral) levels (Fig. 7D ; -0.5% ± 0.2% SD), and remain at half of the control value (-0.5% ± 0.3% SD) at 7 days after induction. The 50% decline in brn 3.2 mRNA correlates well with the later (40%) RGC decrease in rAION-treated retinas (see Fig. 5 ; RGCs in rAION-treated vs. control eyes). Little if any alteration in outer retinal cell function occurred, as measured by the photoreceptor-specific gene opsin mRNA, between induction and 7 days after induction (Fig. 7E ; opsin; 0 days, 1.1% ± 0.2%, and 7 days, -0.2% ± 0.1%). Changes in HSP90 isoform mRNA expression levels are not statistically significant (data not shown). There was a trend for HSP86 (Fig. 7C) and -84 (Fig. 7F) to increase over uninduced retinas from 3 to 7 days (compare Fig. 7C : HSP86, 3–7 days; Fig. 7F : HSP84, 3–7 days). HSP70-1/2 mRNA expression is stable after induction for the first 2 days (1.0% ± 0.2% at 0 days vs. –0.1% ± 0.2% at day 2; Fig. 7B , HSP70-1/2), with a possible slight increase at 3 days after rAION (Fig. 7B ; HSP70-1/2, 3 days, 1.3% ± 0.7%). HSP70-1/2 levels were at baseline 7 days after induction (Fig. 7B ; HSP70-1/2; 7 days, 1.0% ± 0.3%).
|
|
Strong HSP84 protein signal was present in the RGC layer and inner segment of the photoreceptor layer in control retina (Fig. 8G) . Three days after rAION induction, there was an accumulation of HSP84 protein in the NFL (Fig. 8H) . By 7 days after induction, HSP84 signal declined in the NFL and RGC layer (Fig. 8I) . There was increased HSP84 staining in the inner plexiform layer (IPL; Fig. 8I , double arrow), suggesting that reactive changes continue in RGC layers.
|
Discussion |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
After rAION induction, there was a rapid (within 30 minutes) loss of circulation to the ON. rAION-induced edema peaked 1 to 2 days after induction, and resolved by 5 days, whereas human AION-induced ON edema resolves by 4 weeks.20 Despite early resolution of ON edema, ON function was compromised by 3 days after rAION induction, and VEP amplitude remained depressed even 37 days after induction. rAION grossly resulted in a pale, shrunken ON, similar to that seen clinically in human AION.20 The late ON changes were coincident with a loss of 40% of nuclei in the RGC layer. Similar to human AION, rAION specifically altered the number of RGC layer cells without grossly changing the number of cells in the INL and ONL.3 No cellular changes were visibly apparent in the ONL or INL levels 37 days after rAION induction (Fig. 4F) , and there were no quantitative changes in INL or ONL thickness. The rAION-induced cellular losses were largely limited to the RGC layer and are statistically significant. Thus, similar to human AION,20 23 29 rAION produced early functional changes and permanent retinal and ON alterations.
rAION-induced histologic ON changes were apparent 6 days after induction in the rat ON, with axonal swelling and collapse. Myelin sheath breakdown (demyelination) was prominent by day 11. Failure of axonal integrity was thus concurrent with neuronal somatic changes early in the course of rAION. Permanent changes included septal thickening and axonal loss. These changes were most noticeable in the ON center, which represents the vascular "watershed," and is also typical of human AION.2 28 The central loss results suggest that damage in rAION is due to ischemia resulting from preferential damage of the terminal capillaries supplying the nerve center, rather than from a nonspecific effect of dye or thermal toxicity, because the latter two responses would be predicted to have a random effect on axons scattered throughout the ON. The central axonal loss is similar to that reported in many cases of human AION.30 Thrombosis induced by artificially damaging the vascular endothelium, using RB-induced superoxide radicals, could also lead to local effects on RGC axons that are distinct from those caused by natural microvascular occlusion (for example, by release of soluble factors). However, these questions are beyond the scope of the present study.
rAION was used to identify potential retinal gene responses that may be analogous to those occurring in clinical AION. Gene expression results, except for c-fos, though not statistically significant, are suggestive of specific trends. The minimal changes in photoreceptor-specific gene expression suggest that rAION's effects are largely limited to the inner retinal cell layers. The mRNA for c-fos, an immediate-early response gene, through elevated over baseline for the first 3 days after induction, showed two peaks of induction: one at 5 hours after rAION induction, and another at 2 to 3 days. c-Fos expression then declined to baseline 7 days after induction. Strong c-fos expression occurs in other animal models after CNS ischemia, pain, and surgical axotomy,31 32 33 suggesting that some similarities exist in a variety of CNS stress responses. The bimodal response suggests that, similar to brain,33 there may be at least two distinct response periods after rAION-induced retinal stress.
Brn 3.2 mRNA levels rapidly declined, with a plateau 1 day after induction, compared with the uninduced eye, and remained low up to 7 days in rAION retinas. A decline in Brn 3.2 protein was histologically and quantitatively apparent 3 days after induction of rAION. Brn 3.2 expression in the retina is largely restricted to RGCs and is associated with RGC survival, and its loss results in RGC apoptosis.34 The loss of Brn 3.2 activity is explainable by the relative RGC specificity of Brn 3.2 and the decline in RGC-specific functions after rAION. The long-term decrease in retinal expression of Brn 3.2 probably represents RGC functional reprogramming.
There was a trend toward a decline in retinal HSP86 mRNA that was at its maximum by 2 days after induction and returned to normal control levels 3 to 5 days after induction. Although HSP84 mRNA levels also suggest a bimodal retinal response, these changes were small (20%–40% differences) and are not significant. The later increase in HSP86 expression suggests a possible retinal reactive response to RGC axonal ischemia, which is supported by the late (2–3 days) increase in the retinal mRNA levels for the classic stress-response gene HSP70-1/2. Increased RGC-HSP90 expression has also been shown after transient ischemia.35 HSP86 expression is known to increase selectively after stress in a number of other systems, whereas HSP84 is constitutively expressed.36 Although the experimental sample numbers were small, sample trends suggest that retinal gene expression changes occur after rAION and that some of these changes may be biphasic. Biphasic genetic response to traumatic and ischemic CNS injury has been observed.33 Thus, the retina probably modifies its genetic programming rapidly after rAION, to respond to RGC axonal ischemic stress. This hypothesis is supported by the immunochemical changes at the cellular level.
It is evident that there was immunohistological changes in retinal expression of the HSP90 isoform after rAION, although total quantitative HSP90 expression was relatively stable. The latter finding may be explained by the fact that HSP90 proteins are used by all retinal cells, but at relatively different levels and for different functions. HSP90 mRNA and proteins are differentially expressed by RGCs, for axon-intensive functions.18 HSP84 and -86 proteins apparently accumulated in the RGC and NFL layers of the retina up to 3 days after induction. RGC-specific HSP90 isoform expression was considerably reduced at 7 days after rAION. Other (non-RGC) retinal cell types also showed a change in HSP90 expression after rAION. HSP90 isoform signal in control retina was present in the RGC, OPL, and photoreceptors (Prcs). After rAION induction, HSP84- and -86 signal declined at 3 days in the OPL and was reconstituted by 7 days after induction. HSP84 expression may also selectively increase at 3 and 7 days in the INL and IPL. The later changes in HSP90 isoform protein expression in the retina may be a result of non-RGC, stress-associated HSP90 functions that are distinct from their RGC-specific functions. The complex immunohistologic pattern of retinal HSP90 redistribution after ischemic axonopathy, suggests that HSP90 isoforms may perform multiple functions in different retinal cell types.
Our model may differ from the human disorder, in that microvascular occlusion in the model is produced by superoxide radicals, whereas the mechanism of "natural" vascular occlusion in human AION is unknown. Nevertheless, the changes observed after induction of rAION imply that a well-defined sequence of early responses occur after CNS ischemic axonopathy and that irreversible changes may begin within 1 day after axonal ischemia, despite adequate neuron soma vascularization. These early changes probably prime the retina for the later cascade of events related to rAION-induced RGC death. The rAION model can enable a detailed analysis of the individual retina and ON changes occurring after ON stroke, which may be useful in determining possible therapeutic interventions for this disorder.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the V. Kann Rasmussen Foundation (Denmark), a career development award and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness.
Submitted for publication March 17, 2003; revised May 27, 2003; accepted May 29, 2003.
Disclosure: S.L. Bernstein (P); Y. Guo, None; S.E. Kelman, None; R.W. Flower, None; M.A. Johnson, 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: Steven L. Bernstein, Department of Ophthalmology, University of Maryland School of Medicine, 10 S. Pine St. Baltimore, MD 21201; slbernst{at}umaryland.edu.
|
References |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
, and HSP90ß mRNA expression by mitogen activation and heat shock in human lymphocytes Exp Cell Res 192,587-596[CrossRef][Medline][Order article via Infotrieve]This article has been cited by other articles:
|
|
 |
|
  H. F. Edelhauser, J. H. Boatright, J. M. Nickerson, and the Third ARVO/Pfizer Research Institute Working G Drug Delivery to Posterior Intraocular Tissues: Third Annual ARVO/Pfizer Ophthalmics Research Institute Conference Invest. Ophthalmol. Vis. Sci., November 1, 2008; 49(11): 4712 - 4720. [Full Text] [PDF]
|
|
|
|
|
|
B. J. Slater, Z. Mehrabian, Y. Guo, A. Hunter, and S. L. Bernstein Rodent Anterior Ischemic Optic Neuropathy (rAION) Induces Regional Retinal Ganglion Cell Apoptosis with a Unique Temporal Pattern Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3671 - 3676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Chen, M. A. Johnson, R. A. Flower, B. J. Slater, N. R. Miller, and S. L. Bernstein A Primate Model of Nonarteritic Anterior Ischemic Optic Neuropathy Invest. Ophthalmol. Vis. Sci., July 1, 2008; 49(7): 2985 - 2992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Jehle, K. Wingert, C. Dimitriu, W. Meschede, J. Lasseck, M. Bach, and W. A. Lagreze Quantification of Ischemic Damage in the Rat Retina: A Comparative Study Using Evoked Potentials, Electroretinography, and Histology Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1056 - 1064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Bernstein, Y. Guo, B. J. Slater, A. Puche, and S. E. Kelman Neuron Stress and Loss Following Rodent Anterior Ischemic Optic Neuropathy in Double-Reporter Transgenic Mice Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2304 - 2310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. W. Fraunfelder, H. D. Pomeranz, and R. A. Egan Nonarteritic anterior ischemic optic neuropathy and sildenafil. Arch Ophthalmol, May 1, 2006; 124(5): 733 - 734. [Full Text] [PDF]
|
|
|
|
|
|
N. Goldenberg-Cohen, Y. Guo, F. Margolis, Y. Cohen, N. R. Miller, and S. L. Bernstein Oligodendrocyte Dysfunction after Induction of Experimental Anterior Optic Nerve Ischemia Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2716 - 2725. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
