Administration of 17β-Estradiol Attenuates Retinal Ischemia–Reperfusion Injury in Rats

Abstract

purpose. Accumulating evidence has suggested that 17β-estradiol exerts protective effects against ischemic damage in various organs. In addition, leukocytes that accumulate in postischemic tissues are thought to play a central role in ischemia–reperfusion injury. This study was designed to evaluate quantitatively the inhibitory effects of 17β-estradiol on leukocyte accumulation during ischemia–reperfusion injury and on subsequent retinal damage after transient retinal ischemia.

methods. Transient (60 minutes) retinal ischemia was induced in male rats by temporary ligation of the optic nerve. Thirty minutes before induction of ischemia, 17β-estradiol (0.1 mg/kg) was administered intraperitoneally. At 6, 12, 24, and 48 hours after reperfusion, leukocyte accumulation in the retina was evaluated in vivo by means of acridine orange digital fluorography. Histologic and electroretinographic (ERG) studies were carried out to evaluate retinal damage.

results. Treatment with 17β-estradiol significantly inhibited postischemic leukocyte accumulation; the maximum number of accumulating leukocytes was reduced by 35.7% at 24 hours after reperfusion (P = 0.01). Histologic examination showed that administration of 17β-estradiol significantly reduced retinal damage, which was most obvious in the inner retina, 168 hours after reperfusion (P = 0.0001). ERG studies at 12 and 168 hours after reperfusion showed that recovery of the b-wave amplitude was significantly improved with treatment of 17β-estradiol (P = 0.023).

conclusions. The present study demonstrated the inhibitory effects of 17β-estradiol on leukocyte accumulation and subsequent tissue injury during retinal ischemia–reperfusion injury.

Recent investigations have indicated the beneficial effects of estrogen to lower the incidence of ischemic events in various organs, such as ischemic cerebrovascular diseases, ¹ cardiovascular diseases, ² and central retinal vein occlusion. ³ In addition, once patients experience an ischemic insult, it has been suggested that estrogen exerts a protective effect against ischemic organ damage, including that in the cerebrum. ^{4 5} A recent in vivo investigation reported that 17β-estradiol treatment reduced infarct size by approximately 70% in dogs subjected to transient myocardial ischemia. ⁶ However, few data are available about the effects of estrogen on retinal ischemia–reperfusion injury. 

Leukocytes are thought to play a central role in ischemia–reperfusion injury. ^{7 8 9} Leukocytes accumulated in postischemic tissues may cause tissue injury by blocking blood flow, ⁸ producing oxygen free radicals, ^{10 11} and releasing various kinds of inflammatory cytokines. ^{12 13} The importance of leukocytes in ischemia–reperfusion injury has been demonstrated in many experimental studies by showing that prevention of their participation reduces ischemia–reperfusion injury. ^{14 15 16} We demonstrated that inhibition of leukocyte accumulation in the postischemic retina by blocking adhesion molecules reduced retinal damage during retinal ischemia–reperfusion injury. ¹⁷  

Recent studies showed that treatment with 17β-estradiol reduces postischemic leukocyte accumulation and the subsequent ischemic damage after transient myocardial ischemia. ^{18 19} Delyani et al. ¹⁸ have reported that estrogen treatment reduces leukocyte accumulation by approximately 40% in postischemic cardiac tissue. This inhibitory effect of estrogen on leukocyte accumulation after reperfusion may contribute to its protective effect against retinal damage during ischemia–reperfusion injury, but there are presently no reports of such experimental studies. 

We have developed a method of acridine orange digital fluorography that allows us to visualize leukocytes and to evaluate quantitatively leukocyte accumulation in the retina in vivo. ^{20 21 22} Using this technique, we previously evaluated leukocyte accumulation in the rat retina during ischemia–reperfusion injury. ^{17 23} The present study was designed to evaluate quantitatively the inhibitory effects of 17β-estradiol on leukocyte accumulation during retinal ischemia–reperfusion injury in vivo using acridine orange digital fluorography. We also evaluated the protective effect of 17β-estradiol on the subsequent ischemic retinal damage by means of histologic and electroretinographic (ERG) examinations. 

Methods

Ischemia Model

All procedures conformed with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male pigmented Long–Evans rats, weighing 200 to 250 g (n = 116), were anesthetized with a mixture (1:1) of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). Their pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. 

Transient retinal ischemia was induced by the method that has been described previously, ²⁴ with slight modification. ^{13 23} After a lateral conjunctival peritomy, the lateral rectus muscle was disinserted and the optic sheath of the right eye was exposed by careful blunt dissection. A ligature of 6-0 nylon was then placed around the optic sheath and tightened until blood flow in the retinal vessels stopped, as determined by funduscopic examination with an operating microscope. After absence of perfusion for 1 hour, the suture was removed. In the present study, we used only those eyes in which complete reperfusion was confirmed through the operating microscope within 5 minutes of ligature removal. 

To examine whether the administration of 17β-estradiol attenuates retinal ischemia–reperfusion injury, 0.1 mg/kg of 17β-estradiol was given intraperitoneally 30 minutes before induction of ischemia; vehicle-treated rats were given the same volume of saline. 

Acridine Orange Digital Fluorography

Acridine orange digital fluorography has been described previously in detail elsewhere. ^{21 22} This technique uses a scanning laser ophthalmoscope (Rodenstock Instruments, Munich, Germany), coupled with a computer-assisted image analysis system, that makes continuous high-resolution images of fundus stained by acridine orange (Wako Pure Chemicals, Osaka, Japan). Acridine orange, a metachromatic fluorochrome, is a widely used probe in biochemical and cytochemical studies. The dye emits a green fluorescence when it interacts with DNA. The argon blue laser was used for the illumination source, with a regular emission filter for fluorescein angiography because the spectral properties of leukocytes stained with acridine orange are similar to those of sodium fluorescein. 

The evaluation of leukocyte accumulation was performed at 6, 12, 24, and 48 hours after reperfusion in both 17β-estradiol–treated and vehicle-treated groups. Eight eyes of eight different rats were examined at each time point. Eight nonischemic rats were evaluated as the control. In addition, eight rats were given an intraperitoneal injection of 17β-estradiol immediately after reperfusion to examine the effect of 17β-estradiol treatment at the different time points of ischemia on leukocyte accumulation. Immediately before acridine orange digital fluorography, rats were anesthetized with the same agent used before ischemia induction, and the pupils were dilated. A contact lens was placed on the cornea to maintain transparency throughout the experiments. Each rat had a catheter inserted into the tail vein and was placed on a movable platform. Body temperature was maintained between 37°C and 39°C throughout the experiment, and arterial blood pressure was monitored with a blood pressure analyzer (IITC, Woodland Hill, CA; Table 1 ). 

Acridine orange (0.1% solution in saline) was injected continuously through the catheter for 1 minute at a rate of 1 ml/min. At 30 minutes after the injection, the fundus was observed with the scanning laser ophthalmoscope to evaluate leukocytes accumulated in the retinal microcirculation. The obtained images were stored on an S-VHS videotape to allow subsequent quantitative evaluations of leukocyte accumulation. 

The video recordings were analyzed with an image analysis system, as described in detail elsewhere. ^{21 22} In brief, the system consists of a computer equipped with a video digitizer (Radius, San Jose, CA), that digitizes the video image in real time to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. We evaluated the number of leukocytes accumulated in the retinal microcirculation 30 minutes after acridine orange injection. The number of fluorescent dots in the retina within 8 to 10 areas of 100 pixels square at a distance of 1 disc diameter from the edge of the optic disc was counted. The average of the number of dots in the areas studied was used as the number of leukocytes accumulated in the retinal microcirculation for each rat. 

After the described laser ophthalmoscopic images were obtained, the rat was killed with an overdose of anesthesia. The eye was then enucleated to determine a calibration factor to convert values measured on a computer monitor (in pixels) into real values (in micrometers). 

Histologic Analysis

Eight eyes from 8 rats in the 17β-estradiol–treated, vehicle-treated, and nonoperated control groups were obtained to evaluate the severity of retinal damage. After 168 hours of reperfusion, the rats were killed with an overdose of anesthe sia. The operated eyes were immediately enucleated, and a small incision was made at the corneoscleral limbus. These eyes were fixed in 2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer and in 3.7% formaldehyde afterward. The eyes were then dehydrated, embedded in paraffin, sliced with a microtome into 2-μm-thick sections, and stained with hematoxylin and eosin. Each section was cut along the horizontal meridian of the eye through the optic nerve head, perpendicular to the retinal surface. Four retinal sections were examined with an optical microscope (×400) to a masking procedure and then digitized by a charge-coupled device camera on a computer monitor. 

To quantify the degree of retinal damage, we measured changes in thickness and linear cell densities (number of nuclei in a 50-μm-wide band) within the various retinal layers, using the method described by Hughes, ²⁵ with slight modification. ²³ We measured the thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), outer nuclear layer (ONL), and the overall retina from inner to outer limiting membrane (ILM–OLM) and also counted the number of cell nuclei of three retinal layers (ganglion cell layer[ GCL], INL, and ONL). These measurements were made at a distance 1.5 mm from the center of the optic nerve head. The value was averaged from five measurements in the temporal and nasal hemispheres of four different sections, for a total of 40 measurements for each parameter. 

ERG Responses

Six eyes from 6 different rats in the 17β-estradiol–treated and vehicle-treated groups were obtained to evaluate the functional recovery of retinal damage after 12 and 168 hours of reperfusion. With the same agent used before ischemia induction, rats were anesthetized and the pupils were dilated immediately before ERG analysis. Body temperature was maintained at 37C° throughout the experiment. 

After dark adaptation for at least 60 minutes, ERGs were recorded from each eye using a photostimulator lamp placed in front of the eye with a light intensity that was approximately 3.5 × 10⁴ candela (cd)/m² on the surface of the cornea. A carbon electrode (NEC Medical Systems, Tokyo, Japan) was then placed on the cornea and stainless steel needle electrodes (NEC Medical Systems) were placed beneath the skin of the nose and the tail, which served as reference and ground, respectively. The responses were amplified with a time constant of 0.3 second and a high-frequency–cut filter of 1000 Hz (Biological Amplifier 1243; Nihon Kohden, Tokyo, Japan); four trials were averaged with QC-111J (Nihon Kohden). Each b-wave amplitude was measured with a HyperAnalyzer (Kissei Comtec, Tokyo, Japan), and data from three different experiments were averaged for each eye. ERGs were recorded at 12 and 168 hours after reperfusion in both 17β-estradiol–treated and vehicle-treated groups. 

Statistical Analysis

Data are expressed as mean ± SEM. The data were analyzed using ANOVA, with post-hoc comparisons tested using the Fisher’s protected least significant difference test. Differences were considered statistically significant when the probability value was less than 0.05. 

Results

Leukocyte Accumulation in Retinal Microcirculation

After acridine orange injection, leukocytes accumulated in the retina remained fluorescent for approximately 2 hours. At 30 minutes after acridine orange injection, we identified the accumulated leukocytes as distinct fluorescent dots with the highest contrast. The fluorescence of circulating leukocytes decreased gradually after acridine orange injection due to the washout effect and was faint at this time (Fig. 1) . 

Figure 2 shows the time course of the number of leukocytes accumulated in the retinal microcirculation in both vehicle-treated and 17β-estradiol–treated groups. Although few leukocytes could be recognized in control rats, the number of accumulated leukocytes began to increase with time after reperfusion in vehicle-treated rats. The number peaked at 658 ± 32 cells/mm² at 24 hours after reperfusion and decreased thereafter. The number of accumulated leukocytes was significantly less in 17β-estradiol–treated rats than in vehicle-treated rats (P = 0.0025). In 17β-estradiol–treated rats, the number of accumulated leukocytes was 423 ± 26 cells/mm² at 24 hours after reperfusion, which was significantly fewer (by 35.7%; P = 0.01) compared with vehicle-treated rats. 

Figure 3 indicates the effects of 17β-estradiol treatment at the different time points of ischemia on leukocyte accumulation at 24 hours after reperfusion. The number of accumulated leukocytes in rats administered with 17β-estradiol immediately after reperfusion was significantly decreased compared with that in vehicle-treated rats (394 ± 21 cells/mm², P = 0.0068). There was no significant difference between the two groups of rats treated with 17β-estradiol at different time points before ischemia induction and after reperfusion. 

Histologic Analysis

To investigate the protective effect of 17β-estradiol against retinal ischemia–reperfusion injury, we performed a quantitative histologic analysis (Fig. 4) . Our results showed that ischemia–reperfusion injury of the retina caused severe destruction of the inner retinal elements, which resulted in decreased thickness and damage of retinal cells, with less obvious changes in the outer retina, whether the rats were treated with 17β-estradiol or not (Figs. 5 and 6) . The thickness of the IPL, INL, and ILM–OLM in vehicle-treated rats (38.5%, 71.7%, and 82.9% that of nonoperated rats, respectively) was significantly reduced compared with nonoperated rats (P = 0.0001). In addition, cell density of the GCL in vehicle-treated rats (58.6% those of nonoperated rats) was significantly reduced compared with nonoperated rats (P < 0.0001). 

Destruction of the inner retinal elements was significantly ameliorated in 17β-estradiol–treated rats compared with vehicle-treated rats. When treated with 17β-estradiol, thicknesses of the IPL and INL were 68% and 96.4%, respectively, those of nonoperated rats (P = 0.014 and 0.035 compared with vehicle-treated rats). Cell density of the GCL in 17β-estradiol–treated rats was 77.2% that of nonoperated rats (P = 0.0002 compared with vehicle-treated rats). 

Regarding thickness of the ONL and cell density of the INL and ONL, there were no statistically significant differences among the three groups: nonoperated, vehicle-treated, and 17β-estradiol–treated rats. 

ERG Responses

The functional protection of 17β-estradiol against retinal ischemia–reperfusion injury was assessed by ERG analysis (Fig. 7) . In vehicle-treated rats, b-wave recovery was 22.5% and 75.9% of preischemic baseline amplitudes at 12 and 168 hours, respectively, after reperfusion. Administration of 17β-estradiol significantly improved b-wave recovery after ischemia–reperfusion (P = 0.023). b-Wave recovery 12 and 168 hours after reperfusion was 37.7% and 91.0% (P = 0.0055 and 0.046 compared with vehicle-treated rats, respectively) of preischemic baseline amplitudes in 17β-estradiol–treated rats. 

Discussion

Various beneficial effects of estrogen have been of increasing interest among ophthalmologists. Hales et al. ²⁶ reported that estrogen provides protection against cataract formation, whereas Sator et al. ²⁷ demonstrated that estrogen has a positive effect on reduction of intraocular pressure. Miyamoto et al. ²⁸ reported that estrogen inhibits cellular infiltration in endotoxin-induced uveitis. Recently, in organs such as the heart and brain, many studies investigating the benefits of estrogen revealed its protective effects against ischemia–reperfusion injury, ^{4 5 6} although the mechanisms underlying the protective action remain to be determined. So far, no experimental studies have reported the effects of estrogen on retinal ischemia–reperfusion injury. In the present study, we demonstrated that administration of 17β-estradiol significantly reduces leukocyte accumulation after reperfusion and lessens subsequent retinal damage during ischemia–reperfusion injury. 

In this study, we showed that the number of accumulated leukocytes in the retina increased significantly during reperfusion after the transient period of ischemia. Accumulated leukocytes in postischemic tissues have been suggested to be involved in the pathogenesis of ischemia–reperfusion injury ^{7 8 9} by producing oxygen free radicals ^{10 11} and releasing various cytokines. ^{12 13} In addition, Hatchell et al. ⁸ reported capillary plugging by neutrophils in the postischemic rat retina, which was thought to be responsible for the no-reflow phenomenon seen after transient ischemia. Moreover, the deleterious role of leukocytes in ischemia–reperfusion injury has been demonstrated in many experimental studies of various organs, including the cerebrum. ^{14 15 16 29} A recent in vivo study into retinal ischemia–reperfusion injury indicated that reduction of leukocyte accumulation by inhibition of intercellular adhesion molecule (ICAM)-1, one of the adhesion molecules that mediate leukocyte recruitment to the postischemic region, ³⁰ decreased subsequent retinal damage after transient retinal ischemia. ¹⁷  

The present study demonstrated that the administration of 17β-estradiol 30 minutes before ischemia induction inhibited the increase in leukocyte accumulation after reperfusion. The maximal numbers of accumulated leukocytes were reduced by 35.7% with treatment of 17β-estradiol. This inhibitory effect of 17β-estradiol on leukocyte accumulation after reperfusion would contribute to its protective effect against retinal damage during ischemia–reperfusion injury. Similar findings have been reported on the protective effects of 17β-estradiol against myocardial ischemia–reperfusion injury. Several investigators have reported that 17β-estradiol treatment results in a marked reduction of leukocyte infiltration in postischemic myocardium, which leads to less cardiac necrosis. ^{18 19} An experiment by Delyani et al. ¹⁸ showed that administration of 17β-estradiol attenuated leukocyte adherence to coronary vascular endothelium. Squadrito et al. ¹⁹ also showed that the reduced accumulation of leukocytes in estrogen-treated rats was mediated by its inhibitory effect on ICAM-1 expression. Therefore, the inhibitory effect of 17β-estradiol on ICAM-1 expression and subsequent leukocyte adhesion would account for the inhibition of leukocyte accumulation during ischemia–reperfusion injury. 

Our histologic results showed that retinal damage after ischemia–reperfusion injury was most obvious in the inner retina. Previous investigations on retinal damage after ischemia–reperfusion injury have shown that inner retinal elements are morphologically vulnerable to ischemic insults. ^{25 31 32} This specificity might derive in part from the vulnerability of the inner retina to ischemic insult and in part from accumulated leukocytes that initially infiltrate the inner retina. A light microscopic study by Hangai et al. ¹³ showed that neutrophils accumulated in the IPL, GCL, and nerve fiber layer of the postischemic retina, with less conspicuous changes occurring in the INL and ONL. In addition to neural cell death induced by ischemic insult, these accumulated leukocytes would contribute to ischemia–reperfusion injury. Histologic sections in the study described herein showed the protective effect of 17β-estradiol in retinal ischemia–reperfusion injury, especially in the inner retina, and its inhibitory effects on leukocyte accumulation would account for this protective effect. 

In the present study, transient retinal ischemia was induced by temporary optic ligation to make an experimental model of retinal ischemia–reperfusion injury. However, it must be taken into account that ligating the optic nerve may cause transient disturbance of axonal flow, which results in damaging retinal ganglion cells. ³³ It has been demonstrated that axotomy-induced retinal ganglion cell death reaches approximately 40% at 7 days after axotomy. ^{34 35} Therefore, axonal injury by temporal optic ligation may explain our results, which showed that the protective effect of 17β-estradiol on cell density in GCL was moderate, compared with the effect on cell density of INL and thickness of IPL. 

In conclusion, this study is the first to demonstrate that 17β-estradiol protects against postischemic tissue damage of the retina. Considering the role of leukocytes in ischemia–reperfusion injury, our findings suggest that the ability of 17β-estradiol to attenuate leukocyte accumulation after transient ischemia would contribute to this protective effect. 

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

 Submitted for publication July 29, 1999; revised March 6, 2000; accepted March 22, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Mean Arterial Blood Pressure and WBC Count in Peripheral Blood

	Vehicle-Treated							17β-Estradiol–Treated
		6 h	12 h	24 h	48 h	6 h	12 h	24 h	48 h
MABP, mm Hg	85.8 ± 6.1	92.0 ± 7.8	107.0 ± 6.7	102.8 ± 10.2	91.0 ± 5.5	95.3 ± 9.5	111.0 ± 3.3	103.8 ± 5.1
WBC, ×103/μl	7.7 ± 0.6	8.0 ± 1.9	8.6 ± 1.3	9.0 ± 0.7	7.7 ± 0.4	10.0 ± 1.2	9.1 ± 1.8	7.3 ± 1.0

 MABP, mean arterial pressure; WBC, peripheral leukocyte count.

 No significant difference was observed between vehicle-treated rats and 17β-estradiol–treated rats.

Figure 1.

 

Leukocytes accumulated in the retina were observed as fluorescent dots 30 minutes after acridine orange injection. A small number of leukocytes can be found in control rats (A). In vehicle-treated rats, increasing numbers of leukocytes accumulated at 12 (B) and 24 (C) hours after reperfusion. Significant reduction of leukocyte accumulation was seen in 17β-estradiol–treated rats at 12 (D) and 24 (E) hours after reperfusion.

Figure 2.

 

Time course of the number of leukocytes accumulated in the retina after reperfusion in vehicle-treated and 17β-estradiol–treated rats. Values are mean ± SEM. *P < 0.01 compared with vehicle-treated rats.

Figure 3.

 

The number of leukocytes accumulated in the retina 24 hours after reperfusion in rats administered with 17β-estradiol before ischemia induction and after reperfusion. Values are mean ± SEM.* P < 0.01 compared with vehicle-treated rats.

Figure 4.

 

Light micrographs of the retina at a distance of 1.5 mm from the center of the optic nerve head. (A) Nonoperated control rat. (B) Vehicle-treated rat at 168 hours after reperfusion. (C) 17β-estradiol–treated rat at 7 days after reperfusion. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OLM, outer limiting membrane.

Figure 5.

 

Thickness of different retinal layers at 168 hours after reperfusion in vehicle-treated and 17β-estradiol–treated rats. Data are expressed as a percentage of nonoperated control rats. Values are mean ± SEM. *P < 0.01, †P < 0.05 compared with control rats; §P < 0.01,‡ P < 0.05 compared with vehicle-treated rats.

Figure 6.

 

Cell density of the different retinal layers at 168 hours after reperfusion in vehicle-treated and 17β-estradiol–treated rats. Data are expressed as a percentage of nonoperated control rats. Values are mean ± SEM. *P < 0.01 compared with control rats; †P < 0.01 compared with vehicle-treated rats.

Figure 7.

 

Recovery in the b-wave at 12 and 168 hours after reperfusion in vehicle-treated and 17β-estradiol–treated rats. Data are expressed as a percentage of preischemic baseline amplitudes. Values are mean ± SEM. *P < 0.01 compared with vehicle-treated rats.