Published online before print November 1, 2007, doi: 10.1161/STROKEAHA.107.499319
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(Stroke. 2007;38:3237.)
© 2007 American Heart Association, Inc.
Original Contributions |
Novel Mouse Model of Monocular Amaurosis Fugax
From the Centre de Recherche Thérapeutique en Ophtalmologie-CERTO (D.C.L., K.B., E.P., J.L., M.M., M.A.), Faculté de Médecine Paris Descartes; Laboratoire de Génétique Moléculaire (I.B., I.L., M.V.), INSERM U745, Faculté des Sciences Pharmaceutiques et Biologiques; and Service de biostatistiques et d’informatique médicale (J.-P.J.), Faculté de Médecine Paris Descartes, Paris, France.
Correspondence to Marc Abitbol, CERTO, Faculté de Médecine Paris-Descartes, Site Necker, 156 rue de Vaugirard, 75015 Paris, France. E-mail abitbol{at}necker.fr
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Abstract |
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Background and Purpose— Retinal ischemia is a major cause of visual impairment and is associated with a high risk of subsequent ischemic stroke. The retina and its projections are easily accessible for experimental procedures and functional evaluation. We created and characterized a mouse model of global and transient retinal ischemia and provide a comprehensive chronologic profile of some genes that display altered expression during ischemia.
Methods— Ischemia and reperfusion were assessed by observing flat-mounted retinas after systemic fluorescein injection. The temporal pattern of gene expression modulation was evaluated by quantitative reverse transcription–polymerase chain reaction from the occurrence of unilateral 30-minute pterygopalatine artery occlusion until 4 weeks after reperfusion. Electroretinograms evaluated functional sequelae 4 weeks after the ischemic episode and were correlated with histologic lesions.
Results— This model is the first to reproduce the features of transient monocular amaurosis fugax resulting from ophthalmic artery occlusion. The histologic structure was roughly conserved, but functional lesions affected ganglion cells, inner nuclear layer cells, and photoreceptor cells. We observed an early and strong upregulation of c-fos, c-jun, Cox-2, Hsp70, and Gadd34 gene expression and a late decrease in Hsp70 transcript levels.
Conclusions— A murine model of transient retinal ischemia was successfully developed that exhibited the characteristic upregulation of immediate-early genes and persistent functional deficits. The model should prove useful for investigating mechanisms of injury in genetically altered mice and for testing novel neuroprotective drugs.
Key Words: animal models • Gadd34 • heat-shock proteins • retinal ischemia
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Introduction |
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Retinal ischemia is a major cause of visual impairment and blindness. It is involved in various clinical retinal disorders, including ischemic optic neuropathies, obstructive arterial and venous retinopathies, carotid occlusive disorders, retinopathy of prematurity, chronic diabetic retinopathy, and glaucoma.1 Transient visual loss, or amaurosis fugax, is a condition commonly encountered in clinical practice, and its frequent causes include emboli from carotid atherothrombosis or, less frequently, from a cardioembolic source such as mitral valve prolapse. The risk of subsequent hemispheric infarction is increased to
14% within 7 years of an episode of amaurosis. Parallel changes occur in the retina and the brain, even in the absence of traditional risk factors.2,3 The retinotectal system and its projections (which originate from the prosencephalic vesicle) are an excellent model for studying neurodegenerative processes and neuroprotective strategies in the central nervous system, as they are easily accessible for experimental procedures and functional evaluation. In this report, we characterize a new mouse model for global retinal ischemia that reproduces the symptoms of human monocular amaurosis. In our model, we investigated the expression profiles of 6 genes known to be involved in important neuronal ischemic processes from acute ischemia to early and late reperfusion. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the regulation of fibrinolysis4 and in that of N-methyl-D-aspartate receptor–mediated signaling.5 c-jun and c-fos are involved in transcriptional control,6 whereas cyclooxygenase (Cox)-2 is induced during inflammation.7 Gadd34 is a cell cycle protein upregulated in response to DNA damage, cell cycle arrest, and endoplasmic reticulum dysfunction.8 A molecular "chaperone," heat-shock protein (Hsp) 70, also functions as an important cytoprotectant against oxidative stress and apoptosis.9 We also measured the expression levels of 2 control genes, Thy-1 and Rho. These genes encode protein markers specific for 2 types of retinal neurons: ganglion cells (Thy-1) and rod photoreceptors (Rho). Thy-1 mRNA levels provide a sensitive and reliable index of retinal ganglion cell (RGC) injury,10 whereas Rho mRNA levels constitute an index of the global effect of ischemia on rod photoreceptors.11 We investigated the correlation between gene expression profiles and residual functional and histologic retinal lesions 4 weeks after acute retinal ischemia.
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Materials and Methods |
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Animals
Experiments were performed in 8- to 10-week-old male C57BL/6J mice (Charles River Laboratories, Lyon, France). The animals were handled in accordance with European Union and French ethical guidelines for the use of animals in neuroscience research.
Surgery
Animals were anesthetized with 2% isoflurane (Aerrane, Baxter, Maurepas, France) and a mixture of 70% nitrous oxide and 30% oxygen delivered through a close-fitting facemask. Rectal temperatures were maintained at 37±0.5°C. The right common carotid artery was exposed, and the external carotid artery was dissected, ligated, and sectioned to interrupt anastomoses between the ophthalmic artery and the external carotid artery vascular network. The internal carotid artery and its first branch were then dissected, and a silk suture (10-0 Ethilon, Ethicon, Issy-les-Moulineaux, France) was transiently tied around the pterygopalatine artery, which gives rise to the ophthalmic artery. Ischemia was maintained for 15, 30, or 60 minutes, after which the ligature was removed, reperfusion was checked, and the neck incision was closed. Sham-operated animals underwent the same surgical procedure but did not undergo ligation of the pterygopalatine artery (supplemental Figure I, available online at http://stroke.ahajournals.org).12
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Flat-Mounted Retinas
In group A (10 mice), the animals were killed after 30 minutes of acute ischemia to assess the effects of global ischemia induced solely by the surgical procedure and not followed by any reperfusion. Group B (18 mice with 3 animals for each ischemic duration in each group) was used to test the reversal of ischemia at 2 different times after reperfusion (5 minutes and 1 hour) subsequent to the occurrence of 3 distinct ischemic periods (15 minutes for group B15, 30 minutes for group B30, and 60 minutes for group B60). All animals received intracardiac perfusion with fluorescein isothiocyanate (300 µL; Qiagen, Courtaboeuf, France) 2 minutes before being killed. The perfusion pressure was controlled by spontaneous beating of the heart. All flat-mounted retinas were observed with a fluorescence microscope (DMRB, Leica Microsystèmes, Rueil-Malmaison, France). For each animal, both the ischemic right eye and the normally perfused contralateral left eye were removed and fixed by incubation overnight in 4% paraformaldehyde. The cornea and lens were removed. The neural retinas were extracted, flattened by radial incisions, and mounted for further analysis of the macrovasculature and microvasculature. The left eyes served as controls for the quality of the entire procedure, including the evaluation of perfusion pressure.
Quantitative RT-PCR
Forty-two animals (group C) were killed at various times after 30 minutes of retinal ischemia. This ischemic duration was chosen on the basis of literature estimates, indicating that the threshold of the mouse retinal ischemic tolerance ranges between 15 and 30 minutes, and on our preliminary studies demonstrating consistent electroretinogram (ERG) alterations after a 30-minute ischemia duration (data not shown). Seven groups of samples were established, which corresponded to postischemic times of 0 hour, 1 hour, 4 hours, 24 hours, 72 hours, 7 days, and 4 weeks. Six right neuroretinas (3 from mice subjected to ischemia and 3 from sham-operated mice) were collected for RNA extraction for each time point.
The animals were decapitated after brief anesthesia under isoflurane (2%), nitrous oxide (30%), and oxygen (70%). Retinas were rapidly removed, frozen in LN2, and stored at –80°C until RNA extraction. Total RNA was extracted with TRIzol reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s instructions, and quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed as previously described.13 The nucleotide sequences of the primers used for qRT-PCR amplification reactions are available on request.
ERG Recording
Group D (25 mice) was used for functional evaluation of the retinal damage caused by 30 minutes of ischemia, 4 weeks after the surgery procedure, by means of flash ERGs. ERGs were initially recorded 1 week before ischemia to assess the comparability of the ischemic (13 mice) and the sham-operated (12 mice) groups. ERG recordings were then performed on the same animals 4 weeks after ischemia. Three animals (2 ischemic and 1 sham operated) were excluded from the ERG analysis due to hypothermia or recording artifacts. ERG recordings were performed as described elsewhere.14
Histology
Group D mice (13 animals subjected to 30 minutes of ischemia and 12 sham-operated animals) were decapitated under anesthesia after the ERG recordings, as described in the previous paragraph. Right eyes were excised immediately after death, incubated in fixative (4% paraformaldehyde) overnight at 4°C, and embedded in paraffin. Sagittal sections (5 µm) were stained with hematoxylin and eosin. The thickness of each retinal layer (outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, and retinal pigmented epithelium) was measured 150 µm from the center of the optic nerve for the central retina and 300 µm from its extreme edge for the peripheral retina. Measurements were performed on both sides of the optic nerve and on 3 adjacent sections to increase the reliability of the collected data.
Statistical Methods
Comparisons of groups for quantitative data were performed by repeated-measures ANOVA with a mixed-model approach, which takes into account the animal as a random effect and flash intensities and experimental groups as fixed effects.15 All calculations were performed with SAS software, version 8.20 (SAS Institute, Cary, NC). The results are presented as mean±SEM. Values of P<0.05 were considered statistically significant.
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Results |
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The Model Induced Complete and Reversible Retinal Ischemia
On flat-mounted right retinas, ophthalmic artery blood flow and blood resupply from the external carotid artery were completely interrupted in 10 consecutive animals in our model (Figures 1A and 1C). No blood flow was observed from the central retinal artery and its branches vascularizing the inner retina or from the choroidal vascularization ensuring the supply of oxygen and nutrients to the retinal pigmented epithelium and photoreceptor cells. Reperfusion was evaluated 5 minutes and 1 hour after the end of ischemia. It was almost immediate after 15 minutes of ischemia (group B15; Figures 2A and 2B) and was complete at 1 hour (Figures 2C and 2D). Reperfusion was delayed after 30 minutes of ischemia (group B30; Figures 2E and 2F). One hour after ischemia, the B30 group displayed larger-caliber vessels than the B15 group, together with microaneurysms formation and intravascular deposits (Figure 2H). In the B60 group (which underwent 60 minutes of ischemia), reperfusion was just starting at 1 hour (Figures 2K and 2L).
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qRT-PCR Displayed an Early and Transient Increase in Immediate-Early Genes and a Late Decrease in Hsp70 mRNA Levels After Transient Ischemia
The results are summarized in the Table. The PAI1 expression pattern was biphasic, with 1 peak (2.5 times higher than normal) at the end of ischemia and another (3.4-fold increase) 24 hours after reperfusion. The c-jun, c-fos, and Cox-2 mRNA levels showed 8-, 18-, and 5.4-fold increases, respectively, 1 hour after reperfusion; a decrease at 4 hours; and a decline to basal levels within 24 hours of reperfusion. In addition, Cox-2 levels were halved at 72 hours. A transient increase in Gadd34 and Hsp70 mRNA levels (2.8- and 3.4-fold, respectively) was observed 1 hour after reperfusion. The amount of Hsp70, Thy-1, and Rho mRNAs were halved 4 weeks after ischemia.
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Four Weeks After Surgery, 30 Minutes of Ischemia Resulted in a Significant Reduction of the Photopic and Scotopic b-Waves and of the Rod Photoreceptor Waves
The ERG reflects the sum of rod- and cone-mediated retinal responses to light. The a-wave is derived from the photoreceptors. The b-wave results from the interaction of bipolar cells and Müller cells.1 Ischemic and sham-operated mice were similar for all the parameters studied before the intervention. Under scotopic conditions, b-wave amplitude was lower in the ischemic group for all the flash intensities tested. This effect increased with flash intensities up to 10 cds/m2 (20%, P<0.005; Figure 3B). We also observed a significant decrease in a-wave amplitude with flash intensity, of up to 14% for a flash intensity of 10 cds/m2 (P<0.005; Figure 3A). No significant differences were observed between the 2 groups for the a- and b- wave implicit times (data not shown). Under photopic conditions, the response was cone mediated (Figure 4). The b-wave amplitude was significantly smaller (11%, P<0.05 for 10 cds/m2) in the ischemic group. No significant differences between the 2 groups were observed in the photopic a-wave amplitudes or in the photopic a- and b-wave implicit times (data not shown).
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Preservation of Histologic Structure
No significant differences between the ischemic and sham-operated groups were observed in retinal layer thicknesses in the central and peripheral retina (Figure 5 and supplemental Figure II, available online at http://stroke.ahajournals.org).
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Discussion |
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We have designed a purely vascular model of retinal ischemia that reproduces transient human monocular amaurosis. This model is noninvasive with respect to the eye and does not induce blood-eye barrier effraction, mechanical lesions of the retina or optic nerve, contralateral eye lesions, or associated brain lesions in contrast to other currently available models.1 The model is reproducible and easily reversible and involves the vascular structure of the entire eye. Spontaneous reperfusion proceeds progressively from the central retina to the periphery, and its duration increases with the duration of ischemia due to microvascular occlusion. Flat-mounted retinas show microthromboemboli (Figure 2), as observed in acute ischemic rat brains.16 The early and transient upregulation of PAI1 triggers vascular fibrin deposition and contributes to the stabilization and growth of arterial thrombi by abolishing fibrinolysis.4 We observed a decrease in PAI1 mRNA levels 1 hour after reperfusion. The plasminogen activators tissue type (tPA) and urokinase type, which activate the blood fibrinolytic system, are both present in the retina.5 Endogenous tPA potentiates the signaling mediated by glutamatergic receptors, but PAI-1 protein blocks the tPA catalytic site.5 Modification of the PAI1/tPA balance may favor reperfusion but may also increase tPA neurotoxic effects.
Immediate-early genes (c-jun, c-fos, and Cox-2) were strongly induced 1 hour after reperfusion. The proteins c-fos and c-jun are involved in coupling neuronal excitation to target gene expression.6 The associated activation of c-jun and c-fos, as shown by our results, is common during cerebral ischemia17 and has been observed after intravitreal injection of N-methyl-D-aspartate into the rat retina.18 Our model was characterized by a prominent and dramatic increase in c-fos mRNA levels. c-fos is a transcription factor that regulates the cellular mechanisms mediating neuronal excitability and survival.19 However, c-fos expression is also seen in neurons committed to apoptosis.20 The c-jun gene has been linked to neuronal apoptosis21and neuronal rescue.6
Ischemia also upregulates Cox-2 expression; we observed a peak at 1 hour and continued strong expression at 4 hours. Cox-2 reaction products contribute to glutamate excitotoxicity and to the deleterious effects of the inflammatory reaction involving the ischemic brain,7 but Cox-2 activity has also been implicated in the late phase of ischemic preconditioning.22 Cox-2 also plays a protective role in a model of ischemic retinopathy due to an antithrombotic mechanism.23
Gadd34 and Hsp70 are hallmarks of endoplasmic reticulum stress and unfolded protein response.8,24 In our retinal model, a peak in Gadd34 and Hsp70 mRNA levels was observed 1 hour after ischemia. There are reports of Gadd34 overproduction after brain ischemia,25 but there are no reported cases of Gadd34 being detected in the retina. Gadd34 is unstable at both the mRNA and protein level.26 Changes in its expression are short lived in the absence of a positively perpetuating stress signal. As proximal stress sensors are no longer activated, Gadd34 mRNA levels decrease in association with unfolded protein response activation. Gadd34 is associated with cell rescue,25 the restoration of protein synthesis, and DNA repair. It is involved in ischemic preconditioning.27 However, by promoting the resumption of protein synthesis in a cell already burdened by unfolded proteins in the endoplasmic reticulum, Gadd34 may also contribute to cell death.28 Gadd34 is a multifunctional protein and can influence programmed cell death in either a proapoptotic29 or an antiapoptotic8 way, depending on the cell type concerned and the nature and duration of the stress stimulus.
The induced expression of Hsp70 was significant but transient. Little or no constitutive Hsp70 production has been observed in the brain, but Hsp70 is constitutively produced in small amounts in the nuclei of photoreceptors and inner segments.30 These low levels of constitutive Hsp70 production in ocular structures may result from normal levels of light and oxidative stress. The retina has the highest metabolic demand of any tissue in the body. Under normal physiologic conditions and diurnal cycles, the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle decreases in perfusion.31 Nonetheless, the retina displays a remarkable natural resistance to ischemic injury, much greater than that of the brain.1 The induction of Hsp70 production in the brain and retina is associated with cellular resistance to various types of damage.9,32,33
Gadd34 and Hsp70 mRNA levels returned to basal values 24 hours after ischemia, but a second larger peak was observed for PAI1 mRNA. In accordance with our results, Docagne and colleagues34 reported greater PAI1 mRNA levels between 24 hours and 3 days after middle cerebral artery occlusion in mice. The 72-hour stage is characterized by a decrease in Cox-2 gene expression. This minimum could be associated with the resolution of acute inflammatory processes. No prominent upregulation or downregulation of the studied genes was seen 1 week after ischemia.
Low levels of Thy-1, Rho, and Hsp70 seem to be indicators of a dysfunctional retina and may precede histologic cell loss.10 We report evidences of retinal dysfunction in the form of qRT-PCR measurements and ERG recordings. A diminished b-wave is a sensitive marker of ischemic injury, and significant decreases may be observed in tissues with near-normal histology.1 Structural changes are subtler, with only a very slight decrease in the total thickness of all cell layers in the peripheral retina. ERG is therefore a more sensitive indicator of ischemic retinal injury than histologic examination. Furthermore, the use of techniques that measure the panretinal effects of ischemia, such as ERG or qRT-PCR, have the advantage of not being subject to error resulting from nonuniform ischemic retinal changes, whereas histologic analysis may be inadvertently biased by patchy ischemic injury.35 Previous studies that used retrogradely transported tracers to identify the population of RGCs that survived transient ischemia of the retina indicated that RGC loss is an ongoing process that may last up to 3 months after the initial insult.1 Thy1 mRNA abundance and the number of Thy1-expressing cells decreased in advance of detectable RGC loss caused by 3 different modalities of damage. Thus, longer ischemic durations (at least 45 minutes) and/or longer durations for observing animals after ischemia (3 months) may be necessary to detect significant histologic differences in ischemic retinas whatever the methodology used is. This absence of early histologic lesions combined with pure ischemic alterations, which can be easily quantified by sensitive indicators such as ERG and even qRT-PCR late variations, such as Thy-1, Rho, and Hsp70 mRNA modulations, suggest that there might be a window of opportunity for therapeutic intervention in the novel model described in this report.
In conclusion, a murine model of transient retinal ischemia was successfully developed that exhibited the characteristic upregulation of immediate-early genes and persistent functional deficits. The model should prove useful for investigating mechanisms of injury in genetically altered mice and for testing novel neuroprotective drugs.
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Acknowledgments |
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We thank Dr N. Patey-Mariaud and Dr F. Jaubert (Service d’anatomopathologie, Faculté de Médecine Necker-Enfants Malades), Dr C. Marsac (CERTO) for helpful discussions, and E. Le Gall for technical assistance.
Sources of Funding
This work was supported by RETINA-FRANCE and the Ministry for Research of France.
Disclosures
None.
Received July 13, 2007; accepted August 7, 2007.
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