Neuroprotection against Retinal Ischemia–Reperfusion Injury by Blocking the Angiotensin II Type 1 Receptor

Abstract

Purpose.: To investigate the effects of an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II antagonist against retinal ischemia–reperfusion injury in the rat retina.

Methods.: Retinal ischemia was induced by increasing intraocular pressure to 130 mm Hg. Rats were treated with an ACE inhibitor (captopril), an angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), an AT2-R antagonist (PD123319), bradykinin, or a bradykinin B2 receptor antagonist (icatibant). At 7 days after the ischemia, retinal damage was evaluated. Immunohistochemistry and image analysis were used to measure changes in the levels of reactive oxygen species (ROS) and the localization of AT1-R. Dark-adapted full-field electroretinography (ERG) was also performed.

Results.: Pretreatment with captopril or candesartan significantly inhibited the ischemic injury of the inner retina. However, PD123319, bradykinin, or icatibant did not reduce the ischemic damage. In control retinas, retinal vessels were positive for AT1-R. In contrast, 12 hours after ischemia, immunohistochemical analysis detected numerous AT1-R–positive cells in the inner retina in vehicle-treated rats. After ischemia, the production of ROS was detected in retinal cells. However, pretreatment with captopril or candesartan suppressed the production of ROS. On the seventh postoperative day, the amplitudes of the ERG b-waves were significantly lower in the vehicle group than in the groups pretreated with captopril or candesartan.

Conclusions.: The present findings demonstrate that ischemic damage promotes the expression of AT1-R in the inner retina. Both the ACE inhibitor and the AT1-R antagonist that were examined can block the stimulation of the AT1-R and attenuate the subsequent ischemic damage in the rat retina.

The renin-angiotensin system is widely known as a major controller of systemic blood pressure. In this system, angiotensin II (Ang II) plays an essential role in regulating vasomotor tone and ion transport and thus can cause elevation of blood pressure. There are two Ang II receptor subtypes: Ang II type 1 receptor (AT1-R) and Ang II type 2 receptor (AT2-R). ^1–4 Because major Ang II-related systemic functions are mediated by AT1-R signaling, its antagonist action is widely used for the treatment of hypertension and cardiac diseases. Chronic treatments that make use of angiotensin-converting enzyme (ACE) inhibitors or AT1-R antagonists have been reported to reduce stroke incidence and extend the lifespan in stroke-prone spontaneously hypertensive rats (SP-SHRs) ^5,6 and to protect against cerebral ischemic damage in SHRs. ^7,8 In the rat model of endotoxin-induced uveitis, Ang II has been shown to be a promoter of choroidal neovascularization (CNV) and retinal inflammation. ^9–11 Ang II activates the NADPH-dependent oxidase complex, which is a major source of superoxide (O₂ ⁻) and is upregulated in several pathologic conditions associated with oxidative stress. ^12,13 Liu et al. ¹⁴ recently reported that administration of the AT1-R antagonist leads to a protective effect against cerebral ischemia. Moreover, recent evidence suggests that AT2-R may antagonize the action of AT1-R. ¹ AT2-R is expressed in areas related to learning and control of motor activity and is found in fetal tissue. However, it is also present at low levels in adult tissue and is reexpressed in certain pathologic conditions, such as neuronal injury ^15,16 and vascular injury, ¹⁷ suggesting that activation of AT2-R may play a pivotal role in the repair and regeneration of injured tissue. In addition, research is now suggesting possible therapeutic applications for AT2-R, particularly with respect to its protective effects against cerebral ischemia–induced neuronal death. ^18–20  

The kallikrein system (KKS) has been implicated in various pathologic and physiological processes, including inflammation, allergies, blood coagulation, fibrinolysis, and the lowering of systemic blood pressure caused by vessel dilation and diuretic action. ^21,22 Bradykinin, the central molecule of the KKS, is generated by kallikrein from kininogen, including vessel dilation and leakage. ²³ There are at least two types of bradykinin receptors, B1 (B1-R) and B2 (B2-R). ²⁴ B2-R has high affinity for the intact kinin whereas B1-R has greater affinity for the kinin metabolite but weak affinity for the intact kinin. ^24,25 Most of the physiological functions of kinin are mediated by B2-R. ²⁶  

Ischemia-induced injury to the retina, such as diabetic retinopathy and retinal vein occlusion, causes severe and long-lasting visual loss. These morbidities are hard to treat, and research is ongoing regarding possible therapeutic interventions. ^27–31 In addition, many mechanisms of tissue injury-induced ischemia have been proposed. ^32–35 Reactive oxygen species (ROS) trigger ischemic cell damage and lead to the hypersecretion of glutamate and aspartate. ³⁴ An excess amount of glutamate produced under conditions of ischemia–reperfusion stimulates N-methyl-d-aspartate (NMDA), a subtype of the glutamate receptor, ³⁵ and induces an influx of excess Ca^{2 +} into the cell. ^32,33 The purpose of the present study was to investigate the effects of the ACE inhibitor and an Ang II antagonist on neuronal death in retinal ischemia. 

Materials and Methods

Animals

Female Sprague–Dawley rats, each weighing 200 to 250 g, were obtained from Charles River Japan (Yokohama, Japan). Female rats were used because preliminary results indicated no differences between male and female rats (data not shown). Rats were permitted free access to standard rat food (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water. Animal care and all experiments were conducted in accordance with the approved standard guidelines for animal experimentation of the Kagawa University Faculty of Medicine and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Drugs

B2-R antagonist icatibant, AT2-R antagonist (PD123319), and bradykinin were obtained from Sigma-Aldrich (St. Louis, MO); the ACE inhibitor captopril was obtained from Wako (Osaka, Japan); and AT1-R antagonist candesartan was obtained from TRC (North York, Canada). All drugs except candesartan were dissolved in water; candesartan was dissolved in dimethyl sulfoxide (DMSO) to produce stock solutions that were then diluted to the final required concentrations. The final DMSO concentration never exceeded 5%. Drugs were administered intraperitoneally 30 minutes before the induction of ischemia with the exception of PD123319 (5 mg/kg/d), which was administered by subcutaneous osmotic minipump (Alzet model 1007D; Alza Corporation, Mountain View, CA) 1 day before ischemia. The minipumps were implanted subcutaneously into the midscapular region. As the control, animals were pretreated with intraperitoneal injections of vehicle (distilled water or 5% DMSO in PBS) 30 minutes before ischemia. 

Ischemia

Rats were anesthetized with 50 mg/kg pentobarbital sodium (Abbott, Abbott Park, IL) injected intraperitoneally and 0.4% oxybuprocaine hydrochloride administered topically. The anterior chamber of the right eye was cannulated with a 27-gauge infusion needle connected to a reservoir containing normal saline. Intraocular pressure (IOP) was raised to 130 mm Hg for 45 minutes by elevating the saline reservoir. Only the right eye of each rat was subjected to ischemia. Retinal ischemia was indicated by whitening of the iris and fundus. The left eye of each rat served as the nonischemic control. Given that body temperature may influence ischemia-induced retinal ganglion cell death, ³⁶ rectal and tympanic temperatures were maintained at approximately 37°C using a feedback-controlled heating pad (BRC, Nagoya, Japan) during the operation. After restoration of blood flow, temperature continued to be maintained at 37°C. 

Histologic Examination

For the histologic examination, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) 7 days after ischemia and were perfused intracardially with phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde in PBS. Anterior segments, including the lens, were removed. Posterior eyecups were embedded in paraffin, and thin sections (5-μm thickness) were cut using a microtome. Sections were carefully cut to include the full length from superior to inferior along the vertical meridian through the optic nerve head. Each eye was mounted on a silane-coated glass slide and was stained with hematoxylin and eosin. Scleral thickness was measured to confirm that the sections were not oblique. 

Morphometric analysis was performed to quantify ischemic injury. Five sections were selected randomly in each eye. One person with no previous knowledge of the treatments performed all the light microscopic (magnification, 10 × 100; Olympus BX-51, Tokyo, Japan) examinations. A microscopic image of each section within 0.5 to 1 mm superior of the optic disc was scanned. In each computer image, the thickness of the inner plexiform layer (IPL) and inner nuclear layer (INL) was measured. In each eye, the thickness of the IPL + INL was obtained as the mean of four measurements. For each animal, this parameter in the right eye was normalized to that in the intact left eye and was shown as a percentage. 

Retrograde Labeling of Retinal Ganglion Cells

Seven days before kill, hydroxystilbamidine (Molecular Probes Inc., Eugene, OR) was injected bilaterally into the superior colliculi of anesthetized rats. The skull was exposed and kept dry and clean. After identifying and marking the bregma, a small window was drilled in the scalp in both the right and the left hemispheres. The windows were drilled to a depth of 3.6 mm from the surface of the skull and were located 6.8 mm behind the bregma on the anteroposterior axis and 1.5 mm lateral to the midline. Using a Hamilton syringe, 1.5 μL of 2% hydroxystilbamidine was slowly injected into the bilateral superior colliculi. After the skin was sutured over the wound, antibiotic ointment was applied. 

Tissue Preparation and Assessment of RGC Survival

Animals were killed with an overdose of Nembutal at 1 week after 2% hydroxystilbamidine (Molecular Probes Inc.) application. Whole flat-mounted retinas were then assayed for retinal ganglion cell density. Rat eyes were enucleated and fixed in 4% paraformaldehyde for 10 hours at room temperature. After removal of the anterior segments, the resultant posterior eyecups were left in place. Subsequently, four radial cuts were made in the periphery of the eyecup, and the retina was then carefully separated from the retinal pigment epithelium. To prepare the flat mounts, the retina was dissociated from the underlying structures, flattened by making four radial cuts, and spread on a gelatin-coated glass slide. Labeled retinal ganglion cells (RGCs) were visualized under a fluorescence microscope (BX-51/DP70; Olympus) with an ultraviolet filter (blue-violet: 395–440 nm). Fluorescence-labeled RGCs were counted in 12 microscopic fields of retinal tissue from two regions in each quadrant at two different eccentricities, 1 mm (central) and 4 mm (peripheral) away from the optic disc. Software (Image-Pro Plus, version 4.0, Media Cybernetics, Bethesda, MD) was used to count the total number of RGCs in each eye. Changes in the densities of the RGCs were expressed as the RGC survival percentage, which was based on a comparison between the surgical and the contralateral control eyes. The specimens that were compared came from different retinal regions of the same animal. 

Electroretinography

ERG responses were measured after overnight dark adaptation (at least 6 hours) using a recording device (Mayo Corporation, Aichi, Japan) 7 days after ischemia. Pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Santen Pharmaceuticals, Osaka, Japan). All procedures were performed in dim red light, and the rats were kept warm during the procedure. A contact lens electrode was placed on the surface of the cornea. A differential electrode was placed under the skin on the forehead, and a neutral electrode was placed under the skin near the tail. Standard flash ERGs were obtained using a flash intensity of 3 cd · s/m² with a single flash. ERGs were obtained of both eyes for each animal. Ischemic damage to the retina was evaluated as the percentage of the a- and b-wave amplitudes from the right eyes subjected to ischemia compared with the nonischemic left eyes. 

Immunohistochemistry for AT1-R

Eyes were enucleated at 6, 12, or 24 hours after 45 minutes of ischemia. Eyes were then fixed in 4% paraformaldehyde and embedded in paraffin. Retinal sections (5 μm) were rinsed in 100% ethanol twice for 5 minutes each, followed by a separate 95% ethanol and 90% ethanol rinse for 3 minutes each. The sections were then washed using PBS (pH 7.4) three times for 10 minutes each and were treated with 0.3% Triton X-100 in PBS (pH 7.4) for 1 hour After further washing three times for 10 minutes each with PBS (pH 7.4), the sections were blocked in 3% normal horse serum and 1% BSA in PBS for 1 hour to reduce nonspecific labeling. Sections were incubated overnight at 4°C in a 1:100 dilution of rabbit polyclonal antibody against human AT1-R (Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody in PBS containing 0.5% Triton X-100, 5% normal horse serum and 1% BSA. Control sections were prepared by omitting both the primary antibody and the rabbit IgG (1:1000; Vector Laboratories Inc., Burlingame, CA) and incubating only in PBS containing 0.5% Triton X-100, 5% normal horse serum, and 1% BSA overnight at 4°C. After they were washed in PBS for 50 minutes, sections were immersed in alkaline phosphatase (AP; Vectastain ABC-AP Kit; Vector Laboratories Inc.) for 30 minutes at room temperature, washed in PBS for 15 minutes, and processed using the avidin-biotin complex reagent (ABC Kit PK-6101; Vector Laboratories Inc.) for 1 hour at room temperature. Images were acquired using 40× objective lenses (DXM 1200; Nikon, Tokyo, Japan). Image editing software (PhotoShop, version 5.0; Adobe, Mountain View, CA) was used to adjust the brightness and contrast of the images. 

ELISA for AT1-R

Eyes were immediately enucleated 6, 12, and 24 hours after 45 minutes of ischemia, and the retina was carefully isolated. Retinas were put into buffer (IBLysis-I; IBL, Takasaki, Japan) and homogenized. These samples were centrifuged at 10,000 rpm for 10 minutes; the supernatant fluid was then removed and put into each well of a 96-well plate. After 10 μg/mL rabbit anti–AT1-R antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to each well, the plates were covered and kept at 4°C overnight. On the next day, each well was washed with PBST (PBS + 0.05% Tween 20), followed by addition of horseradish peroxidase–conjugated anti–rabbit antibody into each well and further incubation at 4°C for 1 hour. Subsequently, the samples were washed with PBST after which 100 μL substrate (TMBS; IBL) was added, followed by a 30-minute period for development at room temperature in the dark. After the reaction was stopped with 100 μL 1 N H₂SO₄, the OD450 was immediately measured. 

Fluorescence Labeling of ROS

To investigate the production of ROS, we injected 5 mg/kg dihydroethidium (DHE; Sigma-Aldrich) in 5% DMSO in PBS intraperitoneally 15 minutes before ischemia. A 0.3-mL aliquot of distilled water, 1 mg/kg candesartan, or 10 mg/kg captopril was administered intraperitoneally 30 minutes before ischemia. Eyes were enucleated 15 minutes after ischemia and then embedded in the OCT compound (Sakura Finetek, Torrance, CA), after which cryosections (20 μm) were prepared. Sections were examined with a microscope (Radiance 2100/Rainbow; Carl Zeiss, München, Germany) using a laser set (excitation laser 514 nm; emission laser >580 nm). 

Detection of O₂ ⁻ by Formazan Deposition

Detection of O₂ ⁻ by formazan deposition was performed by reduction assay (Nitro Blue Tetrazolium Chloride [NBT]; Wako Chemicals, Tokyo, Japan) with slight modification of the methods of Imai et al. ³⁷ Known amounts of KO₂ (Sigma-Aldrich) were dissolved and diluted with 12 mM dicyclo-hexano-16-crown-6 (crown-6; Sigma-Aldrich) DMSO solution. NBT was also dissolved in 12 mM crown-6 DMSO solution to a final concentration of 0.4 mM (NBT solution). Known amounts of KO₂ solution were added to 1 mL NBT solution, resulting in the immediate formation of O₂ ⁻-reduced NBT and insoluble formazan. The solution was analyzed by spectrophotometer (UM3300; Hitachi, Tokyo, Japan) at a wavelength of 572 nm. After obtaining the calibration curve, O₂ ⁻ in the retina was determined by extraction of the NBT solution. Rats were killed immediately after the experiment, and the retinas were removed as soon as possible. Each retina was immersed in 1 mL NBT solution for 5 minutes, and then the supernatant was analyzed by the spectrophotometer at 572 nm. 

Statistical Analysis

Image analysis was performed (Image-Pro Plus software, version 4.0; Media Cybernetics) to assess the altered ROS reaction area. We used the total red-stained area as the indicator of ROS production. 

All data are presented as the mean ± SD. Data were analyzed using an independent Student's t-test or a Dunnett's multiple comparison test, as appropriate. P < 0.05 was considered statistically significant. 

Results

Histologic Changes in the Retina after Ischemia with Captopril

Figure 1A shows a normal retina with no ischemic procedures. Light microscopic photographs were taken 7 days after ischemia in retinas pretreated with distilled water (Fig. 1B). Significant reduction in the thickness of the IPL + INL to 68.9% ± 18.5% (n = 6) after ischemia was observed. Figure 1C shows the retina 7 days after ischemia in retinas pretreated with captopril (10 mg/kg). In animals pretreated with 0.1, 1, or 10 mg/kg captopril, the thickness of the IPL + INL was 68.6% ± 14.5% (n = 6), 71.6% ± 7.2% (n = 5), and 96.7% ± 23.2% (n = 6) of the control, respectively (Fig. 2). Administration of 10 mg/kg captopril significantly prevented reduction in the thickness of the IPL + INL (P < 0.05). Administration of 10 mg/kg captopril 30 minutes after ischemia also provided a neuroprotective effect (95.2% ± 21.1%, P < 0.05, n = 4; Fig. 2). 

Figure 1.

 

Light micrographs of a cross-section through normal rat retina (A) and 7 days after ischemia without captopril pretreatment (B) or with 10 mg/kg of captopril pretreatment (C). Each microscopic image of the retina was scanned within 0.5 to 1 mm superior of the optic disc. Scale bar, 10 μm.

Figure 2.

 

Percentage change relative to control values in the thickness of the IPL + INL 7 days after ischemia without captopril pretreatment or with 0.1, 1, and 10 mg/kg captopril pretreatment or 10 mg/kg captopril postischemic treatment. Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test). †P < 0.05 versus vehicle (independent Student's t-test).

Effect of Candesartan or PD123319 on the Retina after Ischemia

Figure 3A shows a normal retina with no ischemic procedures. Light microscopic photographs were taken 7 days after ischemia in retinas pretreated with 5% DMSO in PBS (Fig. 3B). Significant reduction in the thickness of the IPL + INL was observed. Figure 3C shows the retina 7 days after ischemia in retinas pretreated with candesartan (1 mg/kg). In animals pretreated with 0.1 or 1 mg/kg candesartan, the thickness of the IPL + INL was 81.2% ± 24.7% (n = 5) or 91.3% ± 23.7% (n = 7) of control, respectively (Fig. 4A). Administration of 1 mg/kg candesartan reduced ischemic damage to the retina (P < 0.05). Administration of 1 mg/kg candesartan 30 minutes after ischemia also provided a neuroprotective effect (89.3% ± 14.6%, P < 0.05, n = 4; Fig. 4A). 

Figure 3.

 

Light micrographs of a cross-section through normal rat retina (A) and 7 days after ischemia without candesartan pretreatment (B) or with 1 mg/kg candesartan pretreatment (C). Each microscopic image of the retina was scanned within 0.5 to 1 mm superior of the optic disc. Scale bar, 10 μm.

Figure 4.

 

Percentage change relative to the control values in the thickness of the IPL + INL 7 days after ischemia without candesartan pretreatment or with 0.1 and 1 mg/kg candesartan pretreatment or 1 mg/kg candesartan postischemic treatment (A) or 5 mg/kg/day PD123319 pretreatment (B). Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test). †P < 0.05 versus vehicle (independent Student's t-test).

A 5-mg/kg/day dose of PD123319 administered subcutaneously by osmotic minipump showed no protective effect against retinal ischemic damage (Fig. 4B). The thickness of the IPL + INL was 78.4% ± 19.4% (P = 0.1; n = 5). 

Effect of Bradykinin or Bradykinin B2 Receptor Antagonist on the Retina after Ischemia

Pretreatment with 1 mg/kg bradykinin (n = 5) or 0.01 to 1 mg/kg icatibant (each dose; n = 5) showed no protective effect against retinal ischemic damage (Fig. 5). However, coinjection of captopril (10 mg/kg) with icatibant (0.1 mg/kg) did have a protective effect against retinal ischemic damage (P < 0.05; n = 3; Fig. 5). Even so, this coinjection of captopril (10 mg/kg) with icatibant (0.1 mg/kg) was not significantly different from the injection of icatibant (0.1 mg/kg) by itself (P = 0.057; independent Student's t-test). 

Figure 5.

 

Percentage change relative to control values in the thickness of the IPL + INL 7 days after ischemia with pretreatment with vehicle; 1 mg/kg bradykinin; 0.01, 0.1, or 1 mg/kg icatibant; or 0.1 mg/kg icatibant coinjected with 10 mg/kg captopril. Data express the mean ± SD. *P < 0.05 versus vehicle (independent Student's t-test).

Effect of Captopril or Candesartan on RGC Survival

Figure 6A shows representative results of the RGC labeling in the vehicle-, captopril-, and candesartan-treated rats. Compared with the vehicle-treated rats, RGC death seemed to be mild in the captopril- and candesartan-treated rats. RGC survival rates in the central retinas of the eyes with ischemia were 53.2% ± 11.3% in the vehicle-treated group (n = 6), 72.9% ± 13.2% in the captopril-treated group (P = 0.02; n = 6), and 71.9% ± 9.9% in the candesartan-treated group (P = 0.03; n = 6; Fig. 6B). In the peripheral retina, RGC survival rates in the eyes with ischemia were 54.2% ± 7.5% in the vehicle-treated group, 73.5% ± 10.9% in the captopril-treated group (P = 0.02), and 75.2% ± 15.4% in the candesartan-treated group (P = 0.01; Fig. 6B). 

Figure 6.

 

Effect of captopril or candesartan on ischemia-induced retinal ganglion cell death. (A) Retrograde labeling of RGCs in nonischemic eyes and 7 days after ischemic injury after administration of vehicle, captopril, or candesartan. Micrographs of the central and peripheral areas were taken approximately 1 and 4 mm from the optic nerve head. Scale bar, 20 μm. (B) RGCs were counted in the central and peripheral areas at approximately 1 and 4 mm from the optic nerve head. Graph depicts the mean ± SD of six animals treated with vehicle, six animals treated with captopril, and six animals treated with candesartan. Data express the mean ± SD of six independent experiments. *P < 0.05 versus vehicle (Dunnett's multiple comparison test).

Effect of Captopril or Candesartan on Retinal Function

Recoveries of a- and b-wave amplitudes are shown in Figure 7. On the seventh postoperative day, a-wave amplitude percentages were 41.6% ± 6.0% in the vehicle-treated group (n = 6), 47.2% ± 8.0% in the captopril-treated group (n = 6), and 43.4% ± 6.5% in the candesartan-treated group (n = 6; Fig. 7A). Percentages for b-wave amplitude were 28.3% ± 4.3%, 38.2% ± 8.0%, and 39.0% ± 6.4%, respectively (Fig. 7B). Recovery rates of b-wave amplitude in the eyes treated with captopril or candesartan were significantly higher than in the vehicle group. There was no significant difference in the recovery rates of the a-wave amplitude either between the captopril group and the vehicle group or between the candesartan group and the vehicle group. Both a- and b-wave amplitudes in the nonischemic eyes were stable and were essentially equal before and after ischemia. 

Figure 7.

 

On postoperative day 7, (A) a-wave amplitude percentages were 41.6% ± 6.0% in the vehicle group, 47.2% ± 8.0% in the captopril group, and 43.4% ± 6.5% in the candesartan group. (B) b-Wave amplitude percentages were 28.3% ± 4.3%, 38.2% ± 4.3%, and 39.0% ± 6.4%, respectively. Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test).

AT1-R Tissue Localization in the Retina after Ischemia

We examined the expression of AT1-R in the retina at 6, 12, and 24 hours after 45 minutes of ischemia (Fig. 8). Figure 8A shows the localization of AT1-R in the normal retina. Although retinal vessels were positive for AT1-R, AT1-R expression was not detected in any layer in the normal retina. However, in the postischemic retina (Figs. 8B–D), immunostaining for AT1-R was detected primarily in the IPL and INL. AT1-R expression increased gradually from the baseline and peaked between 6 and 12 hours after ischemia (Figs. 8C, 8D). 

Figure 8.

 

Immunohistochemical staining of AT1-R expression in the retina. Retinal sections from normal animals (A) or at 6 hours (B), 12 hours (C), or 24 hours (D) after ischemia. Positive staining for AT1-R on the inner retinal vessels. AT1-R was present in the ganglion cell layer and INL of the ischemic retina. Scale bar, 10 μm.

AT1-R Expression in the Retina after Ischemia

Protein levels of AT1-R in the retina were upregulated by ischemia/reperfusion injury (Fig. 9). In the normal retina, the AT1-R protein level was 0.034 ± 0.005 ng/mL (n = 6). However, AT1-R expression peaked between 6 (n = 4) and 12 (n = 4) hours after ischemia and then dramatically decreased by 24 hours after ischemia (n = 4). 

Figure 9.

 

AT1-R expression of normal rat retina and rat retina at 6, 12, and 24 hours after ischemia. Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test).

ROS Activation by Ischemia

We tested whether ROS were suppressed by treatment with 10 mg/kg captopril or 1 mg/kg candesartan. For this purpose, we used DHE staining because DHE specifically reacts with intracellular O₂ ⁻, a ROS, and is converted to the red fluorescent compound ethidium in nuclei. In the postischemic retina, DHE fluorescence was clearly upregulated in retinal neuronal cells, and this up-regulation was efficiently suppressed by captopril or candesartan (Fig. 10A). Figure 10B shows the quantification of the color areas expressed as a percentage change (n = 4 each). Mean ROS activation was significantly suppressed by treatment with captopril or candesartan. 

Figure 10.

 

Effect of captopril or candesartan pretreatment on the release of ROS. (A) ROS, detected by DHE, was upregulated in retinal neuronal cells in the retina after ischemia (vehicle compared with control). However, pretreatment with 10 mg/kg captopril or 1 mg/kg candesartan decreased the level of ROS. Scale bar, 5 μm. (B) Measured area of red (%). Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test).]

Detection of O₂ ⁻ in the Retina after Ischemia

Figure 11 shows the amount of O₂ ⁻ produced after ischemia with pretreatment of vehicle, 10 mg/kg captopril, or 1 mg/kg candesartan. The amount of O₂ ⁻ produced in the retina was 6.73 ± 0.83 μg (control; n = 4), 12.10 ± 1.89 μg (vehicle; n = 4), 7.01 ± 0.44 μg (captopril; n = 4), and 7.68 ± 0.80 μg (candesartan; n = 4). The mean detection of O₂ ⁻ was significantly suppressed by treatment with captopril or candesartan. 

Figure 11.

 

Effect of captopril or candesartan pretreatment on the detection of O₂ ⁻. Data express the mean ± SD. *P < 0.05 versus vehicle (Dunnett's multiple comparison test).

Discussion

We have demonstrated that ischemic injury to the rat retina can be prevented by pretreatment with an ACE inhibitor or an AT1-R antagonist. This result indicates that the local rennin-angiotensin system (RAS) is one of the main pathways of retinal ischemic injury. Furthermore, our results revealed tissue localization of AT1-R upregulation after ischemia in the retina, which further suggests an involvement of the local RAS in ischemic injury and subsequent cell death. 

Glutamate is released from the retina during and after ischemia by raising the IOP. ^29,35,38 Glutamate has been widely known to induce selective damage in the inner layers of the retina. ^34,39 The major causes of cell death after activation of the NMDA subtype of glutamate receptors are the influx of calcium into cells and the generation of free radicals. ⁴⁰ Excessive accumulation of intracellular free Ca^{2 +} ([Ca^{2 +}]_i) can have a wide range of detrimental effects, including inhibition of mitochondrial function, reduction of cellular ATP levels, enhancement of ROS production, and activation of cellular proteases and nitric oxide (NO) synthase. ⁴⁰ In combination, these effects can result in neuronal death. In the present study, we showed that suppression of ischemia-induced ROS production can block the AT1-R, which leads to protection of the neurons from delayed neuronal death. However, it is still unclear whether blocking the AT1-R has any effects on the extracellular glutamate levels or [Ca^{2 +}]_i. Further studies are needed to address why the effect on the RGC and ERG preservation was mild despite the high ROS inhibition observed in this study. 

ACE inhibitors inhibit the formation of Ang II from Ang I, which reduces the action of Ang II at both AT1-R and AT2-R. ACE inhibitors also play another role in the KKS: they prevent the inactivation of bradykinin. Therefore, though administration of an ACE inhibitor decreases Ang II, it also increases bradykinin in the retina. Conversely, AT1-R antagonists act more selectively by blocking the action of Ang II on the AT1-R. The decreased AT1-R–mediated activities of Ang II are the underlying mechanism of the antihypertensive effects of these drugs. 

Bradykinin is well known as a plasma kinin that induces inflammation by way of the B2-R. ²³ B2-R is distributed in rat retinas in situ and in vitro, and bradykinin has a protective effect against neurotoxicity induced by glutamate through B2-R in cultured retinal neurons. ^41,42 However, bradykinin did not have a neuroprotective effect in the present study, possibly because of the different experimental methods used (in vivo vs. in vitro). Several investigators have reported that the B2-R antagonists provide a tissue-protective effect against ischemic injury. ^43,44 In the present study, however, the B2-R antagonist icatibant did not exhibit any protective effect against retinal ischemic damage. Although the major KKS components, which include kininogen, kallikrein, kininase II (ACE), and B2-R, have been shown to be present in the choroid and the retina, ^45,46 KKS was not implicated in the ischemic insult to the retina in the present study. 

A considerable amount of ROS is produced in ischemia, especially during perfusion. The amounts produced are related to the oxygen supply and metabolism; when these are exacerbated, neuronal cell damage occurs. ^29,47 Ang II activates the NADPH-dependent oxidase complex, which serves as a major source of superoxide in addition to upregulation in several pathologic conditions associated with oxidative stress. ^12,13 Given that oxidative stress induces apoptosis in neurons, ⁴⁸ hydrostatic pressure-induced oxidative stress could very well be the mechanism responsible for the similar pressure-induced apoptosis seen in RGC-5 cells ⁴⁹ and animal models. ^50,51 In acute pancreatitis, administration of an AT1-R antagonist in conjunction with losartan can suppress the production of ROS by NADPH oxidase. ⁵² In addition, ACE inhibition or administration of an AT1-R antagonist can reduce oxidative stress and protect dopamine neurons in the 6-hydroxydopamine and MPTP in vivo models of parkinsonism. ^53–55 In the present study, there was an increase in the AT1-R level in the inner retina after the ischemia–reperfusion injury. Therefore, it is conceivable that inhibition of AT1-R could contribute considerably to the observed beneficial effects of captopril or candesartan on the neurologic outcome of ischemia–reperfusion injury. Kurihara et al. ⁵⁶ recently reported that the AT1-R signal caused the production of Ang II in a positive feedback manner in the retina and ultimately promoted inflammation. 

We evaluated functional retinal damage after ischemia–reperfusion injury by measuring the ERG a- and b-wave amplitudes. Although it was impossible to determine the morphologic change in the outer retina with a microscope, Büchi ⁵⁷ showed that cell death in the ONL of rat after ischemia could be observed by electron microscopy. This change in the ONL may be responsible for reducing the a-wave amplitude. The histologic change to the retina after pressure-induced ischemia was curiously irregular in distribution, making it difficult to correlate histologic and electrophysiologic recovery. ⁵⁸ Given that selected regions of the retina (within 0.5–1 mm of the optic disc) were examined histologically, we presume that there is not a good correlation between the ERG b-wave and the histologic results. 

In conclusion, the present study demonstrated that local AT1-R expression is markedly elevated after ischemia–reperfusion injury. Our results provide convincing evidence to suggest that blocking the AT1-R exerts therapeutic effects in cases of retinal ischemic insult. 

Footnotes

 Supported by Grant-in-Aid 20592078 for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Footnotes

 Disclosure: K. Fukuda, None; K. Hirooka, None; M. Mizote, None; T. Nakamura, None; T. Itano, None; F. Shiraga, None