WO2008135536A2

WO2008135536A2 - Non- human animal model for transient or permanent retinal ischemia

Info

Publication number: WO2008135536A2
Application number: PCT/EP2008/055438
Authority: WO
Inventors: Dominique Lelong; Elodie Perez; Maurice Menasche; Marc Mardochée ABITBOL
Original assignee: Universite Rene Descartes - Paris 5; Association Francaise Retinitis Pigmentosa (A.F.R.P.) Aussi Dite Retina France
Priority date: 2007-05-02
Filing date: 2008-05-02
Publication date: 2008-11-13
Also published as: WO2008135536A3

Abstract

The present invention relates to a method for reproducing global transient retinal ischemia or reproducing human transient monocular amaurosis, as well the use of this method for testing neuroprotective drugs.

Description

Methods for Producing Global Transient or Permanent Retinal Ischemia and use thereof for testing Neuroprotective drugs for Treating Human Transient Monocular

Amaurosis and Retinal Strokes

Field of the Invention

The present invention relates to a method for reproducing global transient or permanent retinal ischemia, reproducing transient or permanent human monocular amaurosis, as well the use of this method for testing neuroprotective drugs and drugs promoting synaptic plasticity and recovery.

Background of the Present Invention

Retinal ischemia occurs when the blood supply is not sufficient to support the metabolic demands of the retina. Many cases of visual loss and blindness are caused by diseases in which retinal ischemia is a central feature. These diseases include diabetic retinopathy, retinal vascular occlusion, certain types of glaucoma, ischemic optic neuropathies, obstructive arterial and venous retinopathies, carotid occlusive disorders and retinopathy of prematurity (1 ). Moreover, retinal infarction and transient amaurosis are associated with a high risk of subsequent ischemic cerebral strokes. These ischemic cerebral strokes were found to be mostly large-vessel infarcts in the territory of the ipsilateral carotid artery (2). Retinal arteriolar changes may be a sign of small-artery disease. Parallel changes occur in the retina and the brain, even in the absence of traditional risk factors (3-5).

Amaurosis fugax is a transient monocular visual loss, which may be total or sectorial. It is a blackout of the patient's vision and can occur in isolation or gradually increase in duration. Vision loss can last for only a few seconds or can last for hours. Hence totally different symptoms can occur in patients with amaurosis fugax, which makes the situation for treatment unpredictable. In most cases there are no reliable neurological symptoms or findings to predict if a patient is going to present with amaurosis fugax.

Amaurosis fugax results from either embolic, thrombotic, vasospastic or hematogical phenomenon. Thrombus development occurs via cholesterol deposits and atheroma formation within vessel lumens, which causes transient blood flow cessation. The artheromatus plaque then ulcerates and particles are released which lodge within the vessels resulting in distal ischemia. Alternatively, inflammatory cell infiltration and vascular remodeling of the muscular walls of arteries will lead to the lumen narrowing, and results in amaurosis fugax.

Vasospastic causes of amaurosis can be due to non-embolic idiopathic arterial narrowing or iatrogenic causes due to drugs, surgical procedures, toxicity and collateral effects of treatment, while hematologic causes may be due to polycythemia, anemia, sickle cell disease and hypercoagulable states.

Patients with transient monocular vision experience a "curtain" or "shade coming down over their vision and a dimming, fogging or blurring sensation in the eye.

Treatment will depend on the results of the evaluation by a doctor. If there is evidence of a blockage of more than 70% of the carotid artery surgery may be recommended. If the patient is not a candidate for surgery other treatment such as administration of aspirin and blood thinning agents may be prescribed. Other stroke treatments include thrombolysis, administering neuroprotective drugs to reduce lesions, repair strategies by promoting recovery and synaptic plasticity and use of stem cells. However, there is a need for new neuroprotective drugs that can treat retinal ischemia, amaurosis fugax and retinal stroke. In order to obtain these new neuroprotective drugs models must be created that mimick the diseased state of retinal ischemia or amaurosis fugax.

One of the problems with the models in the art is that they create mechanical optic nerve lesions, by invasive procedures on the eye inducing blood-eye barrier effraction by means of canulations or intraocular injections or concomitant brain infarction. Furthermore, the procedures known in the art can cause contralateral eye lesions or brain lesions as commonly seen secondary to carotid occlusions or hypoxic models.

Well known procedures include raising the intraocular pressure opthalmodynamometrically or chemically by administering saline. Oxygen induced retinopathy is also a procedure that is known and used in the animal models to induce ischemia. However, all of the known models can cause variations in the results since these models vary according to the animal species, the affected tissues, the experimental models themselves, whether the injury is transient or permanent, the duration of ischemia, the timing of the tissue collection after ischemia and evaluation methods used for assessing these animal models. None of the existing experimental models permits relevant comparisons.

It is known that several genes display altered expression during ischemia. PAH (plasminogen activator inhibitor 1 ), the primary regulator of plasminogen activation, has been implicated in the regulation of fibrinolysis (6) and in that of NMDA- receptor-mediated signaling (7). The immediate early genes (IEGs), c-jun, c-fos and cyclooxygenase-2 (Cox-2), are stress-induced genes (8). The AP- 1 components, c- jun and c-fos, are involved in transcriptional control (9), whereas Cox-2, the rate- limiting enzyme in prostaglandin (PG) biosynthesis (10), is induced during inflammation (11 ). Gadd34 (growth arrest and DNA damage-inducible protein) is a cell cycle protein upregulated in response to DNA damage, cell cycle arrest, and endoplasmic reticulum (ER) dysfunction. It promotes the resumption of protein synthesis shut down due to ER stress (12). Hsp70 (heat shock protein 70), originally described as a molecular chaperone, also functions as an important cytoprotectant against oxidative stress and apoptosis (13). Although gene expression of these particular genes was known to be altered during ischemia, it is unclear in the art exactly how the gene expression varied.

Thy-1 and Rho. encode protein markers specific for two types of retinal neuron:, ganglion cells (Thy-1) and rod photoreceptors (Rho) . Thy-1 mRNA levels provide a sensitive and reliable index of retinal ganglion cells (RGC )injury (14, 15), whereas Rho mRNA levels constitute an index of the global effect of ischemia on rod photoreceptors (16).

Therefore, to verify that the model for ischemia is sufficient and repeatable histology may not be sufficient. Hence a correlation between gene expression profiles of Thy-1 and Rho and residual functional and histological retinal lesions would be beneficial to confirm global transient retinal ischemia.

Thus there is a need in the art to provide a model for transient or permanent global ischemia that mimics human monocular amaurosis and can be verified by gene expression and electroretinogram recordings. It is an object of the present invention to provide a method that overcomes the deficiencies in the prior art animal models to create retinal ischemia.

It is an object of the present invention to provide an experimental non-human animal model for producing global retinal ischemia that is non-invasive for the eye, does not induce blood-eye barrier invasion by means of canulations or intraocular injections and does not create mechanical lesions of the retina or optic nerve due to hypertension or surgery.

Yet another object of the present invention is to provide a comprehensive chronological profile of some genes displaying altered expression during ischemia.

It is yet another object of the present invention to provide an ischemic model of the retino-tectal system and its projections (which originate from the prosencephalic vesicle) for studying neurodegenerative processes and neuroprotective strategies in the central nervous system.

It is another object of the present invention to provide a method to reproduce the symptoms of human monocular amaurosis, without mechanical optic nerve lesions, invasion of the eye or concomitant brain infarction.

It is yet another object of the present invention to provide a model for studying retinal strokes.

Yet another object is to provide tools for quantitatively evaluating induced dysfunctions and lesions in the eye.

In yet another object the present invention relates to a method for testing neuroprotective drugs.

These and other objects of the present invention will become apparent from the following description, Examples or preferred embodiments.

SUMMARY OF THE INVENTION The present invention provides a method for reproducing transient global retinal ischemia in a non-human experimental animal, said method comprising:

(a) sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye;

(b) ligating a pterygopalatine artery in said non-human experimental animal using a ligature;

(c) maintaining said ligature in place for a period of time to cause ischemia; and

(d) removing said ligature.

In another aspect the present invention provides a method for reproducing permanent global retinal ischemia in a non-human experimental animal, said method comprising:

(c) maintaining said ligature in place.

In another aspect, a method for producing the symptoms of transient human monocular amaurosis in a non-human experimental animal is provided, said method comprising:

(d)removing said ligature.

In another aspect, method for producing the symptoms of permanent human monocular amaurosis in a non-human experimental animal is provided, said method comprising:

(c) maintaining said ligature in place.

In yet another aspect, the present invention provides a method for testing neuroprotective drugs for transient global retinal ischemia or transient human monocular amaurosis by providing an ischemic non-human experimental animal using the methods described above, administrating a neuroprotective drug to said ischemic non-human experimental animal; and measuring whether said neuroprotective drug treats global retinal ischemia or human monocular amaurosis.

In another aspect the present invention provides a comprehensive chronological profile of some genes displaying altered expression during ischemia. These genes being PAH, c-jun, c-fos, Cox-2, Gadd34 and Hsp70., Thy-1 and Rho.

In yet another aspect the present invention provides a method for triggering endogenous quiescent stem/progenitor cells, stem/progenitor cell division and differentiation in the eyes, said method comprising:

(c) maintaining said ligature in place for a period of time to cause ischemia;

(d)removing said ligature; and

(e) testing for markers of stem/progenitor cells and their progeny. Examples of such markers include pAX6, CHX10, Brdu associated with Ki67, Musashi 1 , DCX, TUJ-1 , MAP2, Neu N, GFAP, Thy-1 , Rho, restin, PKC-α , calbindin and the like by means of immunohistochemistry and in situ hybridization.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 is a drawing illustrating branches of internal carotid artery in relation to the ventral surface of the cranium in the rat (eye vascularization has not been determined precisely in the mouse) (Greene EC, 1935). The origin of the pterygopalatine artery, which is prolonged by the ophthalmic artery is illustrated and used in the method of the present invention to cause ischemia.

Fig. 2 are photographs from fluorescence microscopy experiments of flat- mounted fluorescein-perfused retinas (group A). Right ischemic retinas were compared with left control retinas from the same animal. The surgical procedure interrupted eye vascularization. Whole retinas: scale bar 1 mm Peripheral retinas: scale bar 250 urn

Fig. 3 are photographs from fluorescence microscopy experiments of flat- mounted fluorescein-perfused right retinas at 2 reperfusion times (5 min and 1 hour), following acute ischemia for 15 min (group B15), 30 min (Group B 30) and 60 min (Group B 60). The surgical procedure is reversible. Whole retinas: scale bar 1 mm; Peripheral retinas: scale bar 250 urn

Fig. 4A are bar graphs showing a summary of results (means ± SEM) showing the variation of a-wave amplitude recorded under scotopic conditions with different flash intensities, 4 weeks after 30 min of ischemia (n=11) or sham (n=11) operation. A significant decrease in the a-wave amplitude is observed for all stimulation intensities in the ischemic group and is significantly different (p<0.05*) for flash intensities 1 ; 3; 10 and 25 (respectively p= 0.039, p=0.013, p=0.005, p=0.007).

Fig. 4 B are bar graphs showing a summary of results (means ± SEM) showing the variation in b-wave amplitude recorded under scotopic conditions with different flash intensities, 4 weeks after 30 min of ischemia (n=1 1 ) or sham (n=11 ) operation. A decrease in b-wave amplitude is observed for all stimulation intensities in the ischemic group and is significantly different (p<0.05^*) for flash intensities 0.1 ; 0.3; 1 ; 3; 10 and 25 cds/m² (p=0.038, p=0.013, p=0.005, p= 0.009, p=0.004, p=0.012, respectively).

Fig. 5A are bar graphs illustrating a summary of results (means ± SEM) of the peripheral retina showing the variation of a-wave amplitude recorded under photopic conditions with different flash intensities, 4 weeks after 30 min of ischemia (n= 11 ) or sham (n= 11 ) operation. A slight tendency towards a decrease in amplitude of the a- wave is observed.

Fig. 5B are bar graphs showing a summary of results (means ± SEM) showing the variations in b-wave amplitude recorded under photopic conditions with increasing flash intensities, 4 weeks after 30 min of ischemia (n=1 1 ) or sham (n=11 ) operation, A decrease in b-wave amplitude is observed for all stimulation intensities in the ischemic group and is significantly different (p<0.05*) for flash intensities 10 and 25 cds/m² (p=0.046, p=0.003, respectively).

Fig. 6A and 6B are bar graphs showing the results of the measurements of retinal layer thicknesses four weeks after 30 min of retinal ischemia or sham operation. A: Peripheral retina. A slightly lower total retina thickness, not significant but indicating a potential tendency, was observed between the ischemic group (n=13) and the sham group (n=12) (means ± SEM). B: Central retina. No difference in retinal layer thicknesses (means ± SEM) is observed between the ischemic (n=13) and sham (n=12) groups.

Fig. 7 is a table showing the various genes tested for using RT PCR in the Example 3 and describing alternatives names, their gene function, the NM (nucleotide identification number), and the primers used.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As used herein "ER" means endoplasmic reticulum; "ERG" means electroretinogram; "IEG" refers to immediate early gene; "INL" means inner nuclear layer; "IPL" refers to inner plexiform layer; "ONL" means outer nuclear layer "OPL" means outer plexiform layer; "PE" refers to pigmentary epithelium: "qRT-PCR" means quantitative reverse transcriptase polymerase-chain reaction; "SEM" refers to standard error of the mean.

As used herein "non-human experimental animal" refers to mice, rabbits, rats, guinea pigs, monkeys, cats, pigs, gerbils, dogs and the like.

As used herein "global retinal ischemia" means that loss of blood flow to the entire retina resulting in functional lesions affecting ganglion cells, inner nuclear layer cells and photorecptor cells and hence the vascular structure of the entire eye.

As used herein the word "transient" means reversible ischemia. With respect to the ischemic conditions this time frame is from 5 minutes to 4 days or 5 minutes to 1 hour or 5 minutes up to 4 weeks or 5 minutes up to 24 hours. Thus the ischemic duration can be at least 5 minutes to up to 4 days. The observation of ischemic lesions can be from 5 minutes after the onset of ischemia up to the death of the animal and can vary depending on the age of the animal.

As you herein "permanent ischemia" means nonreversible ischemia in which the ligature is maintained in place.

As used herein, "neuroprotective drugs" means drugs that protect the brain from pathological damage by stopping the retinal cells from dying. Drugs such as calcium channel blockers, gamma-aminobutyric acid (GABA) agonists and modulators, glycine antagonists and modulators, N-methyl-D-aspartate receptor (NMDA) antagonists, α-amino-3-hydroxy-5-methylisoazol-4-propionate (AMPA) and modulators thereof and kaϊnate antagonists and modulators and the like, as well as HSP 70 and chemical inducers of its synthesis or accumulation and Cox-2 inhibitors.

As used herein "transient human monocular amaurosis" means transient monocular blindness.

"Sectioning" as used herein means that there is no collateral blood flow.

It should be recalled at this point that mammalian retina consist of three nuclear layers. The outer layer comprises cell bodies of photoreceptors (rods and cones, whereas the inner layers comprises horizontal, bipolar and amacrine interneurons and Mϋller glia. The innermost layer is the ganglion layer, which consists of ganglion and amacrine neurons. These three cellular layers are separated by outer and inner plexiform layers that are made up of synaptic connections.

More specifically, the present invention relates to a method for reproducing transient global retinal ischemia. This method comprises sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye and ligating the pterygopalatine artery in a non-human experimental animal. The ligature is maintained in place for a period of time to cause ischemia. Any type of ligature can be used to tie off the pterygopalatine artery such as silk, gut, monocryl, monofilament polyamide, polydioxane, vicryl and the like. Clamps can also be used.

Once the pterygopalatine artery is tied off it remains in place for a period of time to cause ischemia. This time period may vary depending on the type of experimental design that is used. Whatever the type of animal model used and depending on the experimental settings ischemia can occur with the ligature in place for at least 5 minutes or 5 minutes to 4 days and the consequences of ischemia can be observed in some instances up to 2 years. After maintaining the ligature in place for a period of time to cause ischemia, the ligature is then removed.

A fluorophore can then be given to the experimental animals in order to test if ischemia was achieved. Any fluorophore can be used such that the ischemia in the retina can be observed by fluorescence microscopy. For example, fluorescein, fluorescein isothiocyanate, sodium fluorescein, indocyanine green and the like.

These fluorophores can be administered intracardiacally by perfusion, intravenously, interperitonally intraarterially and the like. Ischemia is achieved when under fluorescence microscopy of flat mounted retinas no blood flow is observed from the central retinal artery and its branches vascularizing the inner retina or from the choroidal vascularization. This is in comparison with the controls in which blood flow is conserved.

The present invention also relates to a method of reproducing the symptoms of transient human monocular amaurosis in a non-human experimental animal. This method is the same as that for reproducing transient retinal ischemia discussed above and comprises sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye, ligating a pterygopalatine artery using a ligature, maintaining the ligature in place for a period of time to cause ischemia and removing the ligature.

In another aspect methods are provided for reproducing permanent retinal ischemia or reproducing permanent human monocular amaurosis. In this regard the same procedure of sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye, ligating a pterygopalatine artery using a ligature is used but the ligature is maintained in place and is not removed from the non-human experimental animal.

In another aspect the present invention provides a method for testing neuroprotectice drugs for global transient retinal ischemia or transient human monocular amaurosis. This method encompasses generating a non-human experimental animal having ischemia by sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye, ligation of the pterygopalatine artery for a period of time to cause ischemia, administering a neuroprotective drug to the animal and measuring whether said drug treats global retinal ischemia or human monocular amaurosis.

The neuroprotective drug can be administered prior to ischemia or after ischemia is achieved in the non-human experimental animal. Furthermore, angiography on the animal can be performed or visualization of ischemia using a flat- mounted retina technique. Besides these two techniques ERG, PCR and histology can be performed to determine the amount, if any, of ischemia. In this test fluorescein or indocyanine green can be administered to the non-human experimental animal via injection in, for example, the small peritoneum or intravenously, interperitonneally or intraarterially and pictures of the retina are then taken with a fundus camera such as a Topcon camera to determine if ischemia is reduced or treated.

In this regard the measurement of ischemia can be performed using the flat mounted retina technique set forth in the Examples or by recording of electroretinograms and histology.

The neuroprotective drug can be administered in any manner to the experimental animal such as by intravitreal injection, eye drops, subconjunctival injections, periocular injections, topical delivery to the eye, implants and systemic routes. The drug may be mixed to make a liquid, gel or ointment for application directly in the eye with a pharmaceutically acceptable vehicle.

Pharmaceutically acceptable vehicles include carriers, excipients and stabilizers. The formulations can be prepared as set forth in Remington's Pharmaceutical Sciences 16^th edition, Osol A. editor (1980). Examples of carriers, excipients and carriers include saline, PBS, buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid, low molecular weight polypeptides; proteins such as serum albumin, immunoglobulins, gelatins; hydrophilic polymers such as PVP; amino acids of glycine, glutamine, arginine, lysine or asparagines; carbohydrates including such as glucose, mannose or dextrins; sugar alcohols including mannitol or sorbitol; salt-forming counterions such as sodium and/or nonionic surfactants such as Tween®.

Dosages and drug concentrations can be determined depending on the particular use envisioned, as well as the body weight of the mammal. Standard pharmaceutical procedures can be used to determine the toxicity and therapeutic efficacy by using cell cultures or experimental animals. Usually the LD₅₀ (lethal dose to 50% of the population ) and the ED₅₀ (the therapeutically effective dose in 50% of the population) can be determined. The therapeutic index is the dose between the toxic and therapeutic effects. In another aspect the present invention relates to a method for studying retinal strokes and provides tools for quantitatively evaluating induced dysfunctions and lesions. It is well known that blood vessel abnormalities in the retina are associated with "silent strokes." Silent strokes occur when smaller blood vessels in the brain become blocked or ruptured. Retinal microvascularization identifies patients at increased risk of stroke.

In this regard the above described method of creating the ischemic nonhuman experimental animal model by sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye, ligating the pterygopalatine artery for a period of time to cause ischemia then removing the ligation provides an animal model for the study of strokes.

In yet another aspect the present invention provides a method for triggering endogenous quiescent stem/progenitor cells, stem/progenitor cell division and differentiation in the eyes, said method comprising sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye; ligating a pterygopalatine artery in said non-human experimental animal using a ligature; maintaining said ligature in place for a period of time to cause ischemia; removing said ligature; and testing for stem cell markers. Examples of such markers include pAX6, CHX10, BrdU associated with Ki67, Musashi 1 , DCX, TuJ-1 , MAP2, Neu-N, GFAP, Thy-1 , Rho, nestin, PKC-α , calbindin and the like by means of immunohistochemistry and in situ hybridization.

In another aspect the present invention relates to a method for reproducing global transient retinal ischemia or reproducing transient human monocular amaurosis, said method comprising measuring in a retina the gene expression patterns of Thy-1 and Rho is indicative of for global transient retinal ischemia or transient human monocular amaurosis. Other aspects of the invention may become apparent from a study of the Examples below.

METHODS USED IN THE EXAMPLES

Animals

Experiments were performed in eight- to ten-week-old male C57BL/6J&N mice (Charles River Laboratories, Lyon, France) and were approved by the CREEA (ethical committee of Rene Descartes University). The animals were handled in accordance with the ARVO (Association for Research in Vision Ophthalmology) guidelines for the use of animals in ophthalmic and vision research.

Statistical methods

ERG and histological data were recorded and collected by investigators blind to the surgical status of the animals. Comparisons of groups for quantitative data were performed using a mixed model approach considering the animal as a random effect (19). All calculations were performed with SAS software, version 8.20 (SAS Institute, Cary, NC). The results are presented as means ± SEM (standard error of the mean). Values of P<0.05 were considered statistically significant.

EXAMPLES

Example 1 -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°C ± 0.5⁰C. The right common carotid artery was exposed by a midline incision and separation of the omohyoid muscle. The external carotid artery was dissected from the surrounding fascia and nerves, ligated and sectioned. The internal carotid and its first branch were then dissected, and a silk suture (10-0) (Ethilon, Ethicon, lssy Les Moulineaux, France) was transiently tied up around the pterygopalatine artery, the first branch of the internal carotid artery. Ischemia was maintained for 15, 30 or 60 minutes, after which the ligature was removed, reperfusion checked, and the neck incision closed. The animals regained consciousness 5 to 10 minutes after the end of anesthesia and were placed in a cage with access to water and food ad libitum. Sham-operated animals underwent the same surgical procedure, but did not undergo ligation of the pterygo-palatine artery (17). The pterygopalatine artery with respect to the ventral surface of the cranium in the rat is shown in Figure 1.

Example 2- Flat-mounted retina

Group A (10 mice) was constituted to test the existence and reproducibility of complete retinal ischemia in the model. The animals were killed after 30 minutes of ischemia. Group B (18 mice) was used to test the reversal of ischemia (5 min and 1 hour after reperfusion) after undergoing ischemia for various periods (15 min for group B15; 30 min for group B30; 60 min for group B60).

All animals (groups A and B) received a lethal dose of pentobarbital sodium (0.1 ml; Ceva Sante Animale, Liboume, France) and then an intracardiac perfusion with fluorescein isothiocyanate (300 μι_; Qiagen, Courtaboeuf, France) before being killed. The samples were observed with a fluorescence microscope (DMLB, Leica Microsystemes, Rueil-Malmaison, France). The right eye was the ischemic eye; the left eye was the control. The eyes were removed and fixed by incubation overnight in 4% paraformaldehyde, 0.1 M phosphate buffer (PB) pH 7.4 and were then rinsed in PBS (phosphate-buffered saline). The cornea and lens were removed. The neural retinas were extracted, flattened by radial incisions, and mounted (Vectashield, Abcys, Paris, France) for further analysis of the macro and microvascularization.

Vascular fluorescein injection resulted in a holangiotic pattern (therefore a direct blood supply to the entire retinal surface) on the flat-mounted control retinas, as observed in humans, primates and rats (Fig. 2B and 2D). Ophthalmic artery blood flow and blood resupply from the external carotid artery in this model were completely interrupted in 10 consecutive animals, as expected (Fig. 2A and 2C). Thus, this model generated global retinal ischemia. On flat-mounted right retinas, 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 proceeded gradually from the central retina to the peripheral retina (Fig. 3). Reperfusion was almost immediate after 15 minutes of ischemia (group B15) (Fig. 3A and 3B) and was complete at 1 hour (Fig. 3C and 3D). Reperfusion was delayed after 30 minutes of ischemia (group B30) (Fig. 3E and 3F). One hour after ischemia, the B30 group displayed larger caliber vessels than the B15 group, together with microaneurysm formation and intravascular deposits (Fig. 3H). In the B60 group (which underwent 60 min of ischemia), reperfusion was just starting at one hour (Fig. 3K and 3L) and was complete in about two hours (data not shown). Thus the results showed that this model induced complete and reversible retinal ischemia.

Example 3-Quantitative RT-PCR

Forty-two animals (group C) were killed at various times after 30 minutes of retinal ischemia. Seven groups were constituted, corresponding to post-ischemia times of 0 hours, 1 hour, 4 hours, 24 hours, 72 hours, 7 days, and 4 weeks. Six 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 liquid nitrogen and stored at -80⁰C until RNA extraction. Total RNA was extracted using TRIzol® Reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's instructions, and RT-PCR was performed as previously described (18).

The nucleotide sequences of the primers used for PCR amplification were as follows:

PAH- sense: 5' CCACAAGTCTGATGGCAGCACC 3¹ (SEQ I D NO: 1 ), and antisense: 5' CCATCGGGGGTGGTGAACTC 3'(SEQ ID NO:2);

p/W7-sense:5' CACGGCCAACATGCTCAGG 3' (SEQ ID NO:3), and antisense: 5' GCATGAGTTGGCACCCACTGT 3^' (SEQ ID NO:4);

c-fos:sense5' CGGACAGATCTGCGCAAAAGTCCT 3'(SEQ ID NO:5) and

antisense:5'ACTACCATTCCCCAGCCGACTCCT 3'(SEQ ID NO:6); Cox-2-sense:5' GCTCAGCCAGGCAGCAAATC 3'(SEQ ID NO:7), and

antisense:5' TACTGGTCAAATCCTGTGCTCATACA3' (SEQ ID NO:8);

Gadd34- sense:5' TCCTCTAAAAGCTCGGAAGGTACACT3^I(SEQ ID NO:9) and antisense: 5"ATCTCGTGCAAACTGCTCCCA3^r(SEQ ID NO:10);

Hsp 70— sense:? CCCAAGGTGCAGGTGAACTACAA3^I(SEQ ID NO:11) and,

antisense:,5'CCAGGTACGCCTCAGCGATCT 3' (SEQ ID NO:12); r/?y/-^-sense:5'GCTCTCCTGCTCTCAGTCTTGC3'(SEQ ID N0:13), and antisense: 5' CTGGATGGAGTTATCCTTGGTGTT 3' (SEQ ID N0:14);

rhodopsin-sense:5 TCTTTGCCACACTTGGAGGTGAA 3, (SEQ ID NO:15) and antisense:5' CCGAAG CG GAAGTTG CTCATC 3¹ (SEQ ID NO:16)

RPLPO- sense: 5¹ GGCGACCTGGAAGTCCAACT 3'(SEQ ID NO: 17), and

antisense: 5' CATCAG CACCACGGCCTTC 3' (SEQ ID N0:18).

The results are summarized as a relative ratio: mRNA levels in ischemic versus sham-operated mice in Table 1.

Table 1

Sequential expression patterns for the genes studied by qRT-PCR. The numbers indicate relative (ischemic versus sham-operated) expression levels standardized with respect to the internal control (RPLPO). Differences between the groups of a factor of at least two were considered significant. The highly significant results are bolded in this table.

The correlation between gene expression profiles and residual functional and histological retinal lesions after four weeks of acute retinal ischemia was investigated. Thirty minutes of ischemia induced functional alterations in the ischemic retina, in the absence of significant histological changes. These sequelae were concomitant with a decrease in Hsp70 mRNA levels and a late upregulation of Cox-2 mRNA levels.

PAH expression pattern was biphasic, with one 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 an 8-, 18- and 5.4- fold increase, 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 halved at 72 hours and displayed a late (4 weeks) 2-fold increase.

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 mRNA was lower (by a factor of two) four weeks after ischemia. No significant variation of Thy-1 and Rho gene expression was observed during the week following vascular injury, but the mRNA levels for both proteins had halved four weeks after ischemia.

The results showed that qRT-PCR displayed an early and transient increase in IEGs, biphasic expression of Cox-2, and a late decrease in Hsp70 mRNA levels after transient ischemia.

Example 4-Electroretinogram recording

Group D (25 mice) was used for functional evaluation of the retinal damage caused by 30 minutes of ischemia, four weeks after the intervention, by means of flash electroretinograms (ERGs). ERGs were initially recorded one 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, four weeks after ischemia. Three animals (2 ischemic and one sham- operated) were excluded from the ERG analysis due to hypothermia or recording artifacts.

Mice were dark-adapted overnight for each recording and prepared under dim red light. Anesthesia was induced by intramuscular injection of a mixture of ketamine (100 mg/kg; ketamine 1000, Virbac, Carros, France) and xylazine (10 mg/kg; Rompun 2%, Bayer, Puteaux, France). Corneas were anesthetized with a drop of 0.4% oxybuprocaine hydrochloride (Cebesine, Chauvin, Montpellier, France) and pupils were dilated with 0.5% tropicamide (Mydriaticum, Thea, Clermont Ferrand, France) and 10% phenylephrine (Neosynephrine Faure 10%, Pharmaster, Erstein, France) eye drops.

Silver needle electrodes were used. The reference electrode was inserted in the right cheek and the ground electrode in the tail. A gold-wire ring electrode on the cornea was used as the active recording electrode. Hydroxyethylcellulose (Goniosol, Alcon, Rueil-Malmaison, France) was applied to ensure good electrical contact and to keep the cornea moist during the procedure. The body temperature of the animals was monitored and maintained at 37°C ± 0.5°C during the ERG recording. ERGs were unilaterally recorded by a single investigator using a Toennies Multiliner Vision system (Jaeger/Toennies, Hochberg, Germany). The band-pass filter width was between 0.3 and 300 Hz.

Single-flash recordings were obtained both in dark-adapted (scotopic) and light-adapted (photopic) conditions. Single-flash stimuli were presented, with ten increments in intensity (10^"4, 10^"3, 10^~2, 3x10^"2, 10^"\ 3x10^"1, 1 , 3, 10 and 25 cds/m²). Ten responses for each measurement were averaged with an inter-stimulus interval (ISI) of 1.92 seconds or 0.52 Hz (for 10^"4, 10^"3, 10^"2 3x1O^"2, 10^~1 , 3x10-¹ cds/m²), and 15 responses were averaged with an ISI of 4.76 seconds or 0.21 Hz (for 1 and 3 cds/m²) and with an ISI of 9.09 seconds or 0.11 Hz (for 10 and 25 cds/m ). The experiment was carried out in light-adapted conditions and a background illumination of 30 cds/m² was used to abolish rod activity.

The ERG reflects the sum of rod- and cone-mediated retinal responses to light and can therefore be used as an objective and quantitative assessment of visual function. The a-wave is derived from the photoreceptors. The b-wave results from the interaction of ON-bipolar cells and Mϋller cells (1 ). Ischemic and sham- operated mice were similar for all the parameters studied before the intervention. In scotopic conditions, b-wave amplitude was low in the ischemic group for all flash intensities tested. This effect increased with flash intensity up to 10 cds/m² (20%, p<0.005) (Fig. 4B). 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) (Fig. 4A) was also observed. The implicit times of the b-wave were slightly higher (up to 15% higher for an intensity of 10 cds/m²) in the ischemic group, but the differences between the two groups were not significant. No difference was observed between the two groups for the implicit times of the a-wave (data not shown).

In photopic conditions, the response was cone-mediated (Fig. 5). The b-wave amplitude was significantly smaller (3.1 %, p<0.05 for 10 cds/m²) in the ischemic group. No significant differences were observed in the a-wave amplitudes or the implicit times (data not shown) for a- and b-waves between the two groups.

The results showed that four weeks after surgery, 30 minutes of ischemia resulted in a significant reduction of the photopic and scotopic b-waves, and rod photoreceptor waves.

Example 5-Histology

Group D (13 mice subjected to 30 min ischemia and 12 sham-operated mice) mice were decapitated under anesthesia after the ERG recordings, as described above. Right eyes were excised immediately after death, incubated in fixative (4% paraformaldehyde, 0.1 M phosphate buffer (PB: pH 7.4) overnight at 4⁰C and sagittally embedded in paraffin. Sagittal sections (5 μm) were cut along the vertical meridian through the optic nerve, dewaxed, rehydrated, and stained with hematoxylin and eosin.

The stained tissue sections were viewed under a light microscope (Zeiss Axioplan 2, Le Pecq, France) and were digitally photographed (Qimaging Roper Scientific, Evry, France). The thickness of each retinal layer (ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer) 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, for the quantitative assessment of retinal cell loss. Measurements were performed on both sides of the optic nerve and on three adjacent sections to increase the reliability of the collected data.

No differences between the ischemic and the sham-operated groups were observed in retinal layer thicknesses in the central retina . The total peripheral retina thickness of the ischemic group was not significantly smaller than that of the sham- operated group (Fig. 6).

Example 6-Stem/Progenitor Cell Markers

Recent findings indicate the presence of stem cells in the brain throughout life and that neurogenesis is an ongoing process into adulthood (Johnsson et al., 1999, Song et al., 2002). Using several preclinical models of neuronal disease in particular acute ischemic stroke, it has been shown that brain injury, as well as therapeutic regimens can promote neurogenesis beyond the capacity present in naϊve animals (Gould et al., 1997, Jin et al., 2001 , Liu et al., 1998, Snyder et al., 1997, Yoshimura et al., 2001 ). Neural stem cells have also been isolated in the adult retina ( Tropepe et al., 2000 and Temple, 2001 ). Retina stem cells persist in the adult mammalian eye in the ciliary marginal zone. A stem cell potential has also been described for the Mϋller glial cells in the retina (Malatesta et al., 2000 and Fischer and Reh, 2001). This result opens possibilities of regeneration by triggering differentiation of endogenous stem cells in response to ischemic injury. 72 h and 7 days after ischemia was found cells in the ciliary body and ciliary marginal zone co-expressing the retinal progenitor markers Pax6 and Chx10 and a de novo proliferation of cell clusters in the ciliary body revealed by bromodeoxyuridine (BrdU) incorporation onto the DNA. The re- acquisition of embryonic characteristics (Nestin) and expression of the cell cycle entry markers CyclinDI and Ki67 and of mature cell markers (MAP2, TuJ-1 , Thy-1 , Neu-N, RHo, PKCα, calbindin, GFAP) are useful markers in this model.

DISCUSSION

A new and purely vascular model of retinal ischemia that reproduces symptoms of transient human monocular amaurosis has been demonstrated above. This model is non invasive for the eye, and does not induce blood-eye barrier effraction by means of canulations or intraocular injections; there are also no mechanical lesions of the retina or of the optic nerve due to ocular hypertension or surgery, in contrast to other proposed models (1 ). There are no contralateral eye lesions, or no associated brain lesions, as seen secondarily to carotid occlusions or hypoxic models. The model is reproducible and easily reversible, involving the vascular structure of the entire eye. Spontaneous reperfusion proceeds progressively from central retina to the periphery, and its duration increases with the duration of ischemia due to a microvascular occlusion. Flat-mounted retinas show microthromboemboli (Fig. 2), as observed in acute ischemic rat brains (20). The early and transient upregulation of PAH triggers vascular fibrin deposition and contributes to the stabilization and growth of arterial thrombi by abolishing fibrinolysis (6).

A decrease in PAH mRNA levels one hour after reperfusion was observed. Plasminogen activators tPA and uPA, which activate the blood fibrinolytic system, are both present in the retina (7). Endogenous tPA potentiates the signaling mediated by glutamatergic receptors, but PAI-1 protein blocks the tPA catalytic site (7). Modification of the PAI1/tPA balance favors reperfusion, but also increases tPA neurotoxic effects.

IEGs (INTERMEDIATE-EARLY GENES)(OyUn, c-fos, Cox-2) were strongly induced one hour after reperfusion. The proteins c-fos and c-jun are involved in coupling neuronal excitation to target gene expression (21 ) and are the most common components of the activator protein 1 (AP-1 ) transcription factor in mammalian cells (9). Associated activation of c-jun and c-fos, as shown in the results, is common during cerebral ischemia (22) and has been observed after the intravitreal injection of NMDA (N-methyl-D-aspartic acid) into rat retina (23). This model is characterized by a prominent and dramatic increase in c-fos mRNA levels. c-FOS is a transcription factor, regulating the cellular mechanisms mediating neuronal excitability (24-26) and survival (27). However, c-fos expression is also seen in neurons committed to apoptosis (28). The c-jun gene has been linked to neuronal apoptosis (29-31 ) and neuronal rescue (9).

Ischemia upregulates Cox-2 expression and a peak at one hour and continued strong expression at four hours was observed as illustrated in the results in the examples. Cox-2 reaction products contribute to glutamate excitotoxicity, and to the deleterious effects of the inflammatory reaction involving the ischemic brain (32). Inflammation is a key element in the pathological progression of ischemic stroke, in acute conditions, and particularly following reperfusion (33). Cox-2 activity is generally thought to be detrimental, but it has also been implicated in the late phase of ischemic preconditioning. Prostanoids (and their mimetics) attenuate injury and reduce infarct size during myocardial ischemia/reperfusion (34). Cox-2 plays a protective role in a model of ischemic retinopathy due to an antithrombotic mechanism (35),

Gadd34 and HSP70 are hallmarks of ER (endoplasmic reticulum) stress and UPR (unfolded protein response) (12, 36). In this retinal model, a peak in Gadd34 and HSP70 mRNA levels were observed one hour after ischemia. There are reports of Gadd34 overproduction following brain ischemia (37, 38), but there are no reported cases of Gadd34 being detected in the retina. Gadd34 is unstable at both the mRNA and protein levels (39). Changes in its expression are short-lived in the absence of a positively perpetuating stress signal. In contrast to what is observed in the brain, the edematous retina does not secondarily obstruct the microvasculature after reperfusion, as the thin retinal tissue has space (the vitreous cavity) into which it can expand (1 ). As proximal stress sensors are no longer activated, Gadd34 mRNA levels decrease in association with unfolded protein response (UPR) activation. Gadd34 is associated with cell rescue (24, 37, 40) and the restoration of protein synthesis and DNA repair. It is involved in ischemic preconditioning (41 ). However, by promoting the resumption of protein synthesis in a cell already burdened by unfolded proteins in the ER, Gadd34 may also contribute to cell death (42). Gadd34 is a multifacet, multifunctional protein and can influence programmed cell death in a pro-apoptotic (43, 44) or anti-apoptotic (12) 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. This may account for the difference in results reported by Lewden and colleagues (45) and Li and colleagues (46). Lewden and colleagues observed induced Hsp70 overproduction secondary to prolonged (60-90 minutes) retinal ischemia, and Li and colleagues found no consistent change in Hsp70 mRNA or protein levels when evaluating preconditioning in the rat retina. Little or no constitutive Hsp70 production has been observed in the brain, but Hsp70 is constitutively produced in small amounts in the inner segments, the nuclei of the photoreceptors and the outer limiting membrane of the retina (46, 47). These low levels of constitutive Hsp 70 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 physiological 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 (48). 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 (13,49, 50).

The IEGs (Intermediate-Early stage genes), Gadd34 and Hsp70, mRNA levels returned to basal values 24 hours after ischemia, but a second larger peak was observed for PAH mRNA. Docagne and colleagues reported greater PAH mRNA levels between 24 hours and 3 days after middle cerebral artery occlusion in mice (51).

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 up- or downregulation of the studied genes was seen one week after ischemia.

At four weeks, a second peak of Cox-2 mRNA levels was concomitant with the decrease in cell-type markers (7??y7,,R/7o).Cox-2-generated PGE2 is known to regulate membrane excitability and long-term synaptic plasticity (52). It is unclear whether the late induction of Cox-2 is detrimental or beneficial.

Low levels of Thy-1 , Rho and Hsp70 appear to be indicators of a dysfunctional retina and precede histological cell loss. Evidence of retinal dysfunction in the form of qRT-PCR measurements and ERG recordings is seen in the results. A diminished b-wave is a sensitive marker of ischemic injury and significant decreases may be observed in tissues with near-normal histology (1 ). In scotopic conditions, the rod- mediated components dominate the response (with pure rod responses at lower luminances and the cone system increasingly contributing at higher luminances). The major differences in the scotopic a-wave amplitude between ischemic and sham-operated animal groups concerned higher luminances. Like the slight decrease in photopic a-wave amplitude, this indicates cone injury in addition to rod injury. This change was not significant, but the small proportion of cones in the mouse retina (3% of all photoreceptors) (53) and the relatively short duration of ischemia make it difficult to assess these variations. Cones may also be intrinsically more resistant to ischemia than rods, and/or may require less energy.

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 histological examination. Furthermore, the use of techniques measuring the panretinal effect of ischemia, such as ERG or qRT-PCR have the advantage of not being subject to error resulting from nonuniform ischemic retinal changes, whereas histologicai analysis may be inadvertently biased by patchy ischemic injury (54),

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Claims

What is claimed is:

1.A method for reproducing global transient retinal ischemia in a non-human experimental animal, said method comprising:

(c)maintaining said ligature in place for a period of time to cause ischemia; and

(d)removing said ligature.

2. The method according to Claim 1 , wherein said time period for causing ischemia is at least 5 minutes or between 15 and 60 minutes or between 5 minutes and 4 days.

3. The method according to Claim 1 , wherein said pterygopalatine artery is ligated with a silk suture.

4. The method according to Claim 1 , further comprising after step (c) perfusing a fluorphore intracardiacally into said non-human experimental animal.

5. The method according to Claim 4, wherein said fluorophore is selected from the group indocyanine green and fluorescein isothiocyanate.

6. The method according to Claim 5, wherein said fluorophore is fluorescein isothiocyanate.

7. The method according to Claim 1 , wherein said non-human experimental animal is selected from the group of mice, rabbits, rats, guinea pigs, monkeys, cats, pigs and gerbils.

8. A method for reproducing the symptoms of transient human monocular amaurosis in a non-human experimental animal, said method comprising:

a.) sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye; b.) ligating a pterygopalatine artery in said non-human experimental animal using a ligature; c.) maintaining said ligature in place for a period of time to cause ischemia; and d.) removing said ligature.

9. The method according to Claim 8, wherein said time period for causing ischemia is at least 5 minutes or between 15 and 60 minutes or between 5 minutes and 4 days.

10. The method according to Claim 8, wherein said pterygopalatine artery is ligated with a silk suture.

11. The method according to Claim 8, further comprising after step (c) perfusing a fluorphore intracardiacally into said non-human experimental animal.

12. The method according to Claim 11 , wherein said fluorophore is selected from the group of idocyanine green and fluorescein isothiocyanate

13. The method according to Claim 12, wherein said fluorophore is fluorescein isothiocyanate.

14. The method according to Claim 8, wherein said non-human experimental animal is selected from the group of mice, rabbits, rats, guinea pigs, monkeys, cats, pigs, dogs and gerbils.

15. A method for testing neuroprotective drugs for transient global retinal ischemia or human monocular amaurosis said method comprising:

a.) sectioning an external carotid artery and all the vascular branches susceptible to provide any blood supply or resupply to the eye;

b.) ligating a pterygopalatine artery in a non-human experimental animal using a ligature;

c.) maintaining said ligature in place for a period of time to cause ischemia;

d.)removing said ligature;

e.) administrating a neuroprotective drug to said non-human experimental animal;

f.) measuring whether said neuroprotective drug treats global retinal ischemia or human monocular amaurosis.

16. The method according to Claim 15, wherein said measuring step (e) comprises administering a fluorophore to said non-human experimental animal and measuring the ischemia using fluorescent microscopy.

17. The method according to Claim 15, wherein said measuring step (e) comprising quantitative reverse transcription PCR wherein c-fos, c-jun, Gadd34, Hsp70, PAH, Thy-1 , Rho and Cox-2 gene expression is measured.

18. The method according to Claim 15, where said measuring step (e) comprising recording electroretinograms.

19. A method for testing for global retinal ischemia or human monocular amaurosis, said method comprising measuring in a retina the gene expression patterns of Thy-1 and Rho.

20. The method according to Claim 19, wherein said gene expression patterns are measured using quantitative reverse transcription PCR.

21. A method for triggering endogenous quiescent stem/progenitor cells, stem/progenitor cell division and differentiation in the eyes, said method comprising:

(c) maintaining said ligature in place for a period of time to cause ischemia;

(d)removing said ligature; and

(e) testing for stem/progenitor cell markers and their progeny.

22. The method according to Claim 21 , wherein stem/progenitor cell markers are selected from pAX6, CHX10, BrdU associated with Ki67, Musashi 1 , DCX, TuJ-1 , MAP2, Neu-N, GFAP, Thy-1 , Rho, nestin, PKC-α and calbindin.

23. A method for reproducing permanent global retinal ischemia in a non-human experimental animal, said method comprising:

(b) ligating a pterygo-palatine artery in said non-human experimental animal using a ligature;

(c)maintaining said ligature in place.

24. A method for producing the symptoms of permanent human monocular amaurosis in a non-human experimental animal is provided, said method comprising:

(a) sectioning an external carotid artery and its downstream anastomosis from an ophthalmic artery;

(c) maintaining said ligature in place.