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(Investigative Ophthalmology and Visual Science. 2000;41:2743-2747.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Transient Ischemic Injury in the Rat Retina Caused by Thrombotic Occlusion–Thrombolytic Reperfusion

Lina Daugeliene1,2, Masayuki Niwa1, Akira Hara3, Hiroyuki Matsuno1, Tetsuya Yamamoto2, Yoshiaki Kitazawa2 and Toshihiko Uematsu1

From the Departments of 1 Pharmacology, 2 Ophthalmology, and 3 Pathology, Gifu University School of Medicine, Japan.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To establish a clinically relevant model of transient retinal ischemia by thrombotic occlusion-thrombolytic reperfusion of the central retinal artery of the rat.

METHODS. Thrombus was photochemically induced in the central retinal artery by the combination of intravenous injection of photo-sensitive dye, rose bengal, and green laser irradiation focused on the artery. Transient retinal ischemia for 60 minutes was achieved by a subsequent systemic administration of tissue-type plasminogen activator to reperfuse the occluded vessel. Samples of retinas were excised from the animals killed 3, 9, 12, 24, 48, and 78 hours after the reperfusion. The experimental data were processed using the TdT-dUTP terminal nick-end labeling (TUNEL) method to detect apoptotic cells.

RESULTS. The transient retinal ischemia caused time-sequential apoptotic changes in the retinal cells as evaluated by counting the number of TUNEL-positive cells. The most remarkable changes occurred in the central area of retina, and further on the sections taken 24 hours after reperfusion. The peripheral area was less affected, and the outer nuclear cell layer was almost unaffected throughout the observation period.

CONCLUSIONS. The proposed method to cause retinal transient ischemia is highly reproducible, and it is easy to simulate the progress and topographical distribution of retinal changes observed in the clinical cases of central retinal arterial occlusion and its subsequent thrombolytic reperfusion. This may provide a useful tool for constructing the effective thrombolytic strategies against the central retinal arterial occlusion and for evaluating the effects of neuroprotective agents.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Several recent studies have revealed that transient retinal ischemia could induce retinal damage. In such studies, transient ischemia of the retina was achieved by using either of two ischemic insults, by increasing intraocular pressure (IOP) above the arterial blood pressure1 2 3 or by ligating the optic nerve together with the central retinal artery for some period.2 However, the former insult may cause some mechanical damage on the neuronal cells due to the high pressure itself, whereas the latter may cause some indirect influences on them due to stagnant neurotrophic transports through the ligated nerve bundles in addition to a possible ligation of collateral blood supplies.

Rose bengal is an iodinated fluorescein dye that efficiently initiates a photochemical reaction, releasing singlet molecular oxygen when irradiated by green light.4 Systemic intravenous injection of rose bengal and its subsequent photoactivation by green light from the outside of the target vessel facilitates the damage to vascular endothelium by liberated active oxygen, which subsequently activates platelets, leading to a formation of transluminal thrombus localized at the irradiated part of the vessel.5 Recently, the combination of photosensitive dye and light illumination has been applied to produce diffuse microthrombi in retinal and choroidal vessels or to form thrombus in some larger branches of central retinal artery or vein and to examine the subsequent morphologic characteristics of retinal injury due to ischemia.6 7 8 9

Thrombolytic therapy with tissue-type plasminogen activator (tPA) is established to be highly effective for recanalization of the coronary or other vessels obstructed by intraluminal thrombi.5 10 To experimentally obtain a transient ischemia of some tissues, photochemically occluded vessels can be reperfused with the use of tPA. However, the model of transient retinal ischemia achieved by thrombotic occlusion-thrombolytic reperfusion of the central retinal artery, which may be encountered in clinical settings, has not been reported yet.

A new noninvasive and clinically relevant model of transient retinal ischemia has been developed. The technique is based on formation of intraluminal thrombus in the rat central retinal artery with the use of a combination of intravenously administered rose bengal and green laser irradiation of the vessel, and by subsequently dissolving the thrombus by means of intravenous injection of tPA. Neuronal cell death occurred in the retina after the transient ischemia. Neuronal damage was time-sequentially followed and histochemically examined using the TdT-dUTP terminal nick-end labeling (TUNEL) staining technique. Thus, this model is expected to be quite similar to the central retinal artery occlusion (CRAO) in patients and useful for investigating the mechanism of retinal injuries due to transient ischemia and evaluating the pharmacological strategies to treat or prevent them.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
Adult male Wistar rats weighing 300 to 350 g were obtained from a local breeder. The animals were handled in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Experimental Protocol
After a rat was anesthetized with an intraperitoneal injection of 6% sodium pentobarbital (0.1 ml/100 g body wt), a small plastic catheter was inserted into the femoral vein for injecting rose bengal (Sigma, St. Louis, MO), dissolved in 0.9% saline at a concentration of 20 mg/ml. The dye was injected (20 mg/kg) just before light exposure. The right eye was subjected to ischemia, and the contralateral eye served as a control.

The topical anesthetic of 0.4% oxybuprocaine (Benoxyl; Santen Pharmaceutical, Osaka, Japan) was applied to the eye. Pupils were dilated with an eyedrop of 0.5% phenylephrine hydrochloride–0.5% tropicamide (Mydrin P; Santen Pharmaceutical). A one-mirror goniolens filled with ethylcellulose (Scopisol; Senju Pharmaceutical, Osaka, Japan) was placed on a rat’s eye for obtaining clear fundus view. The central retinal artery was irradiated with argon green laser (wavelength, 514 nm) using an argon-dye laser apparatus (model ADC-8000; Nidek, Gamagori, Japan). After the injection of rose bengal as bolus, the laser beam was focused on the photosensitized central retinal artery and irradiated for 0.3 second. The laser settings were as follows: 50 µm of the laser diameter and 0.1 W of the laser power.

A total of 38 rats was subjected to thrombolytic reperfusion of the occluded central retinal artery by tPA, which was started to be administered through the catheter placed in the femoral vein 60 minutes after thrombus formation. A half of total tPA (0.27 mg/kg body wt) was first injected as bolus. Then, the remaining half was further infused within 10 minutes. After the administration of tPA the reperfusion was observed in all rats. The fundus was observed ophthalmoscopically 1, 3, and 24 hours after the reperfusion and just before the rats were killed.

Histologic Examination
Under a deep anesthesia with sodium pentobarbital, animals were fixed by intracardiac perfusion of 4% paraformaldehyde phosphate-buffered saline buffer 3 (n = 6), 9 (n = 6), 24 (n = 8), 48 (n = 6), and 72 (n = 6) hours after a blood flow in the retina was restored. The eyes were enucleated and left overnight at 4°C. Then the specimens were embedded in paraffin. Sagittal sections were obtained through the optic nerve. The sections were 4 µm thick, and at the interval of at least 100 µm. Sections were stained with hematoxylin and eosin and were examined using light microscopy.

The TUNEL was performed to detect internucleosomal DNA fragmentation. The staining was performed as described elsewhere.11 After incubation with 20 µg/ml protein kinase K (Sigma), the sections were immersed in TDT buffer (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). TDT (Boehringer–Mannheim GmbH, Mannheim, Germany) and biotinylated dUTP (Boehringer–Mannheim GmbH) were diluted in TDT buffer at a concentration of 125 eu/ml and 12.5 nmol/ml, respectively. The solution was placed on the sections, and then the sections were incubated at 37°C for 60 minutes. The sections were covered with streptavidin peroxidase (Dako, Carpinteria, CA) and stained with 3,3'-diaminobenzidine as a substrate for peroxidase. Finally, counterstaining was done using Mayer’s hematoxylin.

The numbers of labeled cells in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) were counted from six oblong areas, representing parapapillary, central, and peripheral areas of the retina, approximately 1-mm length each, and chosen from both sides of the optic nerve head, in sections obtained at each time point. Data were analyzed independently by two coauthors (LD, MN) in a blinded fashion. The TUNEL-positive cells were averaged and plotted as a number of TUNEL-positive cells per 1 mm length of the area for each retinal layer. Confocal microscope was used for the measurements. The experimental results are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Formation of Thrombus in the Central Retinal Artery
A thrombus was induced in the central retinal artery by the combination of rose bengal and green laser. Retinal arteries became empty, followed by emptying of the veins (Fig. 1B) . To confirm that these changes were caused by the thrombus formation not by spasm in the central retinal artery, tPA (0.27 mg/kg body wt) was intravenously injected as a bolus to dissolve the thrombus 5 minutes after irradiation. Vessels were reperfused immediately after the tPA injection, showing rather hyperemic filling of the vessels (Fig. 1C) . The photographs of the above-mentioned time-sequential changes in the ocular fundus are illustrated in Figure 1 . Without the artificial reperfusion with tPA, the retina became edematous around 1 hour after irradiation. No spontaneous vascular recanalization was observed 24 and 48 hours later after irradiation. The samples of retinas taken 1 week after ischemia showed marked degeneration of the inner retina, although periphery of the retina was almost intact.



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  		Figure 1. Photographs of the central retinal vasculature: before argon green laser irradiation (A); after irradiation (B); and during injection of the tPA (C).

 
Achievement of Transient Ischemia in the Retina
During the administration of the first half dose of tPA (0.135 mg/kg) at 60 minutes after the thrombotic occlusion, blood circulation was noticed in retinal arteries. The blood circulation was supported by continuing the infusion of tPA within 10 minutes in all 38 rats and persisted until they were killed in 32 animals (84%). The retinal edema spontaneously decreased 3 hours later.

Histologic Changes in the Retina
Light-microscopic observation of the 3-hour postischemic samples revealed slightly edematous inner plexiform layer. Appearance of pyknotic nuclei, vacuolated spaces, and degenerative changes in the GCL and INL were noticed especially at 24 hours after ischemia. The 72-hour specimens showed a slight decrease in the cell number of the inner retina. No alterations were seen in the photoreceptor layer. Also, the retinal periphery looked intact.

No TUNEL-positive cells were found in the control eyes. Distribution of TUNEL-positive cells in GCL, INL, and ONL of parapapillary, central, and peripheral retinal areas are shown in Table 1 and Figure 2 . The GCL at 3 hours after ischemia showed the earliest appearance of TUNEL-positive cells; the peak was reached at 9 hours after ischemia induction. TUNEL-positive cells in INL appeared in the animal group killed 9 hours after induction of ischemia. The highest level of the positive cells was in the 24-hour group. TUNEL-positive cells in ONL appeared in the 24-hour samples, and the peak was reached at 48 hours after ischemia induction. The distribution of TUNEL-positive cells is not equal among different retinal areas and layers. The most prominent retinal damage developed in the parapapillary and central areas of inner retina. The periphery and ONL showed minimal changes.


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  		Table 1. Distribution of TUNEL-Positive Cells

 


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  		Figure 2. TUNEL staining of retinas at 24 hours after reperfusion. (A) Normal control, parapapillary area. (B) Normal control, central area. (C) Normal control, peripheral area. (D) Reperfused eye, parapapillary area. (E) Reperfused eye, central area. (F) Reperfused eye, peripheral area. TUNEL-positive cells are seen in GCL and INL. Magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We were able to create a highly reproducible transient ischemia of the rat retina by photochemically induced thrombotic occlusion of the central retinal artery and its subsequent thrombolytic reperfusion with the use of tPA. After the transient retinal ischemia for 60 minutes induced by this method, TUNEL-positive cells were observed in the retina, as identified by TUNEL staining. The results have a different chronological course and different topographical distribution within the retina compared with the conventional transient ischemia inducing methods (i.e., optic nerve ligation and elevated IOP).1 2 3 With our method the parapapillar and central areas of the retina were affected to the highest degree; and the peripheral area and the outer cell layer were rather preserved, whereas the peripheral area is reported to be equally affected with the conventional methods, like optic nerve ligation or increased IOP, and the changes in outer layer are significant, although delayed. This might be explained by considering that a high IOP equally affects every part of the retina because of spherical shape of the eye, and the optic nerve ligation might ligate the collateral vessels together with the central retinal artery and, in addition, strangulate neurotrophic transports through the nerves leading to damage of every part of the retina. Therefore, the transient retinal ischemia induced by our method is considered to be clinically relevant, representing, for example, the CRAO and its subsequent reperfusion by the treatment with tPA.

Actually, the inner layer neurons are significantly more susceptible to ischemia compared with the outer layer neurons12 as shown in the present study. This selective vulnerability could be attributed to different responses between the inner and outer neuronal layers to neurotransmitter, free radical, or rate of protein, like p53, synthesis.1 2 3 Our results are almost identical regarding time and extent of neuronal damage in the inner retina to those found in the transient ischemic model.2 The ONL was found to be almost intact. The reason for this is not only a different blood supply from that in the inner retina but also due to the fact that ONL neuronal cells are less sensitive to ischemic damage. The peripheral retinal area located near the orra serrata was affected to a minimum degree. It might be associated with different blood supply.

Based on the increasing experience and success of fibrinolytic therapy in cerebral, coronary, and other arterial occlusions, attempts have been made to use selective fibrinolysis also in treating acute CRAO.13 There were some reports indicating that locally administered urokinase-type plasminogen activator (uPA) introduced through a microcatheter into the ophthalmic artery within 6 hours after the onset of clinical symptoms is more effective in improving visual acuity than conventional therapies.14 As in the case of stroke, where a systemic fibrinolysis with tPA within 3 hours turned out to be beneficial,15 systemic administration of tPA rather than uPA may be potentially useful for CRAO.

In fact, for reperfusion we used recombinant tPA, which is now preferred clinically as a thrombolytic agent because it has a specific affinity to plasminogen within the established thrombus and generates plasmin, leading to fibrinolysis only at the site of thrombus with less incidence of bleeding as opposed to uPA. In addition, owing to a high affinity of tPA to thrombus, systemic intravenous administration is applicable.

Recently, it has been reported that tPA itself increases neuronal damage after focal cerebral ischemia16 because tPA-deficient mice were resistant to neuronal degeneration due to glutamate analogue administration or to focal cerebral ischemia. The reports claimed that tPA promotes desirable (thrombolytic) as well as undesirable (neurotoxic) outcomes during stroke, representing a two-edged sword. In the present study, we needed a rather large dose of tPA to reperfuse the rather small artery of the rat at 60 minutes after occlusion, a rather long period. However, the number of TUNEL-positive cells itself was comparable to those in the reports using either ligation or elevated IOP methods. In addition, the neuronal cells were clearly preserved in the more peripheral and more outer areas of the retina compared with the other methods.

The ischemic time was found to be a very critical issue for retinal damage severity. A successful restoration of blood flow and improved visual fields were reported in two cases where administration of tPA took place 7 hours after the onset of retinal artery occlusion.13 According to some studies, 60-minute ischemia showed extensive pathologic changes rather than 45-minute ischemia.17 Thus, the earlier blood flow restoration can result in better visual acuity preservation.

An occlusion similar to those observed in the clinical settings could be photochemically induced in the central retinal artery of the rat, and its highly reproducible dissolution could be achieved by systemic tPA injection. This model could be applied for a quantitative analysis of pathologic changes especially in evaluating neuroprotective interventions. Therefore, the present model of central retinal thrombosis may serve as a useful tool for evaluating the efficiency of antithrombotic and/or thrombolytic agents as well as combined thrombolytic therapy with neuroprotectants.


   Footnotes
 
Supported in part by the Grant-in-Aid for Scientific Research (No. 12470015) from the Japanese Ministry of Education, Science, Sports and Culture.

Submitted for publication December 9, 1999; revised March 21, 2000; accepted April 11, 2000.

Commercial relationships policy: N.

Corresponding author: Lina Daugeliene, Department of Ophthalmology, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan. lina-gif{at}umin.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Joo, CK, Choi, JS, Ko, HW, et al (1999) Necrosis and apoptosis after retinal ischemia: involvement of NMDA-mediated excitotoxicity and p53 Invest Ophthalmol Vis Sci 40,713-720[Abstract/Free Full Text]
  2. Kuroiwa, S, Katai, N, Shibuki, H, et al (1998) Expression of cell cycle-related genes in dying cells in retinal ischemic injury Invest Ophthalmol Vis Sci 39,610-617[Abstract/Free Full Text]
  3. Perlman, JI, McCole, SM, Pulluru, P, Chang, CJ, Lam, TT, Tso, MOM (1996) Disturbances in the distribution of neurotransmitters in the rat retina after ischemia Curr Eye Res 15,589-596[Medline][Order article via Infotrieve]
  4. Kusama, Y, Bernier, M, Hearse, DJ (1989) Singlet oxygen-induced arrhythmias: dose-response and light-response studies for photoactivation of rose bengal in the rat heart Circulation 80,1432-1448[Abstract/Free Full Text]
  5. Matsuno, H, Uematsu, T, Nagashima, S, Nakashima, M. (1991) Photochemically induced thrombosis model in rat femoral artery and evaluation of effects of heparin and tissue-type plasminogen activator with use of this model J Pharmacol Methods 25,303-318[Medline][Order article via Infotrieve]
  6. Moroni, F, Lombardi, G, Pellegrini–Faussone, S, Moroni, F. (1993) Photochemically-induced lesion of the rat retina: a quantitative model for the evaluation of ischemia-induced retinal damage Vision Res 33,1887-1891[Medline][Order article via Infotrieve]
  7. Büchi, ER, Szczesny, PJ (1996) Necrosis and apoptosis in neuroretina and pigment epithelium after diffuse photodynamic action in rats: a light and electron microscopic study Jpn J Ophthalmol 40,1-11[Medline][Order article via Infotrieve]
  8. Mosinger, JL, Olney, JW (1989) Photothrombosis-induced ischemic neuronal degeneration in the rat retina Exp Neurol 105,110-113[Medline][Order article via Infotrieve]
  9. Saito, Y, Park, L, Skolik, SA, et al (1997) Experimental preretinal neovascularization by laser-induced venous thrombosis in rats Curr Eye Res 16,26-33[Medline][Order article via Infotrieve]
  10. Collen, D, Topol, EJ, Tiefenbrun, AJ, et al (1984) Coronary thrombolysis with recombinant human tissue-type plasminogen activator: a prospective, randomized placebo-control trial Circulation 70,1012-1017[Abstract/Free Full Text]
  11. Hara, A, Niwa, M, Iwai, T, et al (1999) Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing Brain Res Protoc 4,140-146[Medline][Order article via Infotrieve]
  12. Hayreh, SS, Weingeist, TA (1980) Experimental occlusion of the central artery of the retina, IV: retinal tolerance time to acute ischemia Br J Ophthalmol 64,818-825[Abstract/Free Full Text]
  13. Bertram, B, Wolf, S, Fisches, H, Schulte, K, Hoberg, A, Reim, M. (1991) Lysebehandlung bei retinalen Arterienverschlussen mit Plasminogen-Aktivator Klinische Monatsblatter fur Augenheilkunde 198(4),295-300[Medline][Order article via Infotrieve]
  14. Weber, J, Remonda, L, Heinrich, PM, et al (1998) Selective intra-arterial fibrinolysis of acute central retinal artery occlusion Stroke 29,2076-2079[Abstract/Free Full Text]
  15. . The NINDS rt-PA Stroke Study Group (1995) Tissue plasminogen activator for acute ischemic stroke N Engl J Med 333,1581-1587[Abstract/Free Full Text]
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