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Blood-retinal barrier breakdown following experimental retinal ischemia and reperfusion
Exp. Eye Res. (19"95) 61, 547-557
Blood-Retinal Barrier Breakdown Following Experimental Retinal Ischemia and Reperfusion C H A R L E S A . W l L S O N a*, B R U C E A . BERKOWlTZa, b,L H I D E H A R U F U N A T S U ~.d, D A V I D C..M'ETRIKINL D A V I D W . H A R R I S O N " , M I C H A E L K . LAMa AND PETER L. S O N K I N " Departments of aOphthalmology, bRadiology, and cBiomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX, U.S.A. and ~Department of Ophthalmology, Tokyo Women's Medical College, Tokyo, Japan ,(Received Houston 25 January 1995 and accepted in revised form 6 June 1995) The purpose of this study was to examine the effects of acute ischemia and reperfusion on blood-retinal barrier (BRB) function in the rabbit eye. Hydrostatic pressure (140 mmHg) was used to create total retinal ischemia for intervals of 20, 40, 60, 80 or 100 min in the rabbit eye, The location, size and permeability-surface-area product normalized to the area of retinal leakage (PS') of ischemia-induced BRB lesions were then measured using contrast-enhanced magnetic resonance imaging (MRI) after various intervals of reperfusion. Diffuse outer BRB leakage occurred in most eyes subjected to 80 or 100 min of ischemia. A posterior region of outer BRB sparing was found in eyes that underwent lesser durations of ischemia. On day 1 after retinal ischemia, a linear relationship was found between mean PS' and duration of ischemia for periods of ischemia between 20 and 100 min [slope: 5.65 x 10 -6 to 5.96 × 10 -G cm min 1 (min ischemia)-l; r 2 ~> 0-69]. Lesion size also increased between 20 and 1013min of ischemia. In a longitudinal study, eyes exposed to 60 rain of ischemia showed a decrease in PS and a lesion size over an 8-week period of observation. However, leakage was still present on post-ischemia day 57 in two of three eyes examined. Data obtained in these experiments are expected to prove useful in future studies aimed at understanding how BRB damage relates to neuroretinal damage after ischemia-reperfusion injury. © 1995 Academic Press Limited Key words: blood-retinal barrier, ischemia-reperfusion, rabbit, magnetic resonance imaging.
1. Introduction Ischemia is a component of m a n y retinal and choroidal vascular diseases. While acute retinal ischemia can cause direct cellular injury or death (Foulds and Johnson, 1974: Marmor and Dalai, 1993; Kaskel et al., 1976; Johnson and Foulds, 1978), the relative contribution of post-ischemic sequelae, such as disruption of the blood-retinal barrier (BRB), to retinal damage is less certain. In theory, ischemia sufficient to cause severe injury to the retinal vascular endothelium (inner BRB) a n d / o r retinal pigment epithelium (outer BRB) would be expected to cause influx of bloodderived water and solutes (exudate) into the retina (Cunha-Vaz, 1979), ultimately leading to permanent structural damage (Ferris and Patz, 1984). In addition, migration of exudate outside the primary area of BRB injury could result in increased visual morbidity, such as often occurs in diabetic macular edema (Ferris and Patz, 1984). Knowledge of the effects of ischemia on retinal structure and function has been furthered by use of animal models. For experimental purposes, retinal and choroidal ischemia is commonly achieved by elevating
* For correspondence at: Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9057, U.S.A.
0014-4835/95/110547+ 11 $12.00/0
the intraocular pressure until ocular blood flow is stopped. The histologic and electroretinographic effects of this procedure on the retina have been described previously in several species (Heike and Marmor, 1991 ; Foulds and Johnson, 1974; Marmor and Dalai, 1993; McKechnie, Johnson and Foulds, 1982; Johnson and Foulds, 1978). However, the effect of pressure-ischemia on blood-retinal barrier integrity has not been investigated in detail. In this study, contrast-enhanced magnetic resonance imaging (MRI) was used to quantitate the effects of ischemia on BRB breakdown in the rabbit eye. As has been shown in previous studies (Berkowitz et al., 1991, 1992), the MRI procedure provides a means of localizing leakage as well as measuring BRB permeability and lesion size. Such studies are facilitated by available MRI contrast agents that are freely diffusable and similar to fluorescein dye in molecular weight. Several biologic questions are addressed in this study. The first set of questions extends from a previous report of the light microscopic findings in the pressureischemic rabbit eye (Marmor and Dalai, 1993). The report indicated that neuroretinal and retinal pigment epithelial damage had occurred in a non-uniform or ' p a t c h y ' fashion. From this information, two questions were posed: does pressure-ischemia damage the BRB uniformly or is the damage non-uniform as suggested © 1995 Academic Press Limited
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by the morphologic data ? and, if non-uniform, what portions of the BRB are most susceptible to ischemic injury ? Another issue in the rabbit model relates to tolerance to ischemia. Hayreh and colleagues observed a threshold duration of ischemia that would result in irreversible retinal damage in the pressure-ischemic primate eye (Hayreh, Kolder and Weingeist, 1980). Yet, electroretinographic studies in the rabbit and cat eye have suggested a graded response to increasing duration of ischemia (Foulds and Johnson, 1974; Ulrich and Reimann, 1986). This raises the following questions regarding the behavior of the BRB: does a graded relationship exist between duration of ischemia and the severity of BRB breakdown ? Secondly, is there a threshold duration of ischemia that separates complete BRB recovery from acute damage ? In addition to the above considerations, the issue of cellular proliferation raises yet other questions. Injured retinal vascular endothelial and retinal pigment epithelial cells may remain capable of proliferation and, presumably, restoration of function. Therefore, the present study sought to determine whether, to what degree, and how rapidly BRB function is restored after ischemic damage in the rabbit model. 2. Materials and Methods
Animals and Anaesthesia Nineteen Dutch-belted (pigmented) rabbits weighing 1.9 to 2.0 kg were used in these experiments. Studies were conducted in accordance with institutional guidelines. Anaesthesia was induced by intramuscular injection of ketamine HC1 (25 mg kg -1) and xylazine HC1 (5 mgkg -1) and was maintained by intravenous infusion of ketamine HCI ( 4 0 m g k g - l h r -1) and xylazine ( 4 m g k g lhr-1) administered through a marginal ear vein. Pupils were dilated prior to ocular procedures with tropicamide HC1 (1%) and phenylephrine HC1 (2.5 %) eye drops.
Production of Ischemia A 20-gauge intravenous infusion catheter was placed in the anterior chamber at the limbus and secured with a 6-0 silk purse-string suture. The catheter was connected via intravenous infusion tubing to a bag of lactated Ringer's solution. The amount of pressure elevation at the level of the eye was adjusted by varying the height of the bag. During ischemia, bag height was adjusted to create a pressure of 140 mmHg at eye level; the pressure was confirmed using a calibrated transducer. Although arterial blood pressures were not monitored in these animals during ischemia, previous and subsequent experiments under similar conditions have revealed that systolic arterial pressures (measured from the auricular artery) range from 80 mmHg to 110 mmHg. Indirect ophthalmo-
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scopy was performed at the start of ischemia and periodically thereafter to confirm the absence of blood flow in the retinal blood vessels at the optic disk and along the medullary rays. In one animal, 0.2 ml of 10% sodium fluorescein was injected intravenously immediately after induction of ischemia. Fluorescein ophthalmoscopy showed no dye appearance in the eye until the pressure was released. After a given period of ischemia (see experimental protocols below), the pressure was lowered by removal of the anterior chamber catheter. In all eyes, indirect ophthalmoscopy revealed that blood flow was restored to the preretinal vasculature following catheter removal. In the fluorescein-injected animal, dye rapidly filled the anterior chamber upon removal of the catheter indicating that the anterior segment was reperfused. Corneo-scleral wounds were sutured closed using 8-0 polyglactin. An antibiotic ointment containing polymyxin B sulfate (10000 U g 1) and bacitracin zinc (500 U g i) was applied to the cornea at the end of the operative procedure.
Experimental Protocols In the first experiment, eyes were subjected to varying durations (20, 40, 60, 80 or 100 min) of ischemia and were examined using contrast-enhanced MRI 1 day later. Control eyes did not undergo ischemia, In one control eye, an anterior chamber catheter was inserted and later removed to control for the effects of surgical trauma. Twelve animals were divided into three groups of four animals. Their eyes were used in the experiment as follows: group 1 underwent 20 rain and (fellow eye) 49 min of ischemia, group 2 underwent 60 min and (fellow eye) 80 rain of ischemia, and group 3 underwent 100 min of ischemia and (fellow eye) no ischemia. Groups 1 and 2 were killed by intravenous KC1 injection immediately following the imaging procedure. Three of the animals in group 3 were killed on postischemia day 58, and the eyes were salvaged for histologic examination (see histology below). The fourth animal in this group died of suspected complications of anesthesia. In a second experiment, seven rabbits were subjected to a 60-min period of ischemia in one eye. These animals were then examined using MRI on postischemia days 1, 3, 8, 15, 29 and 57. Three of the seven animals completed all days of imaging. The others died during the follow-up period of suspected complications of anesthesia. On post-ischemia day 58, surviving animals were killed and their eyes salvaged for histologic examination (see histology below).
Magnetic Resonance Imaging During MRI, each animal was anesthetized as described above and mechanically ventilated via an endotracheal tube. The auricular artery was cannu-
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luted from which the heart rate, blood pressure and arterial blood gases were monitored. Ventilatory settings were adjusted to maintain the arterial blood gases to within a physiologically normal range (pH 7"35-7"45; PO 2 8 0 - 1 2 0 mmHg; PCO 2 2 0 - 4 0 mmHg). A warming pad was used to maintain core body temperature. MRI was performed on a 0.5T Toshiba clinical system (Toshiba America MRI, South San Francisco, CA, U.S.A.) using a 10 cm surface coil). Axial T1weighted images (TR = 400 ms and TE = 15 ms) with a slice thickness of 3"5 m m were obtained through the geometric center of both globes. These slices were oriented perpendicular to the long axis of the vascularized medullary ray. Each image required 6.9 rain to acquire. A control image was obtained followed by a bolus intravenous injection of contrast (Gd-DTPA; 1"0 mmol kg-1). Three sequential postinjection images were then acquired.
Image Analysis Images were transferred to a Macintosh llci data station and analysed using the program Image (version 1.50; W. Rasband, National Institutes of Health). A user-defined subroutine facilitated calculation of the permeability-surface area product normalized to the surface area of leaky retina (PS') according to previously published methods (Berkowitz et al., 1992). This measurement involved defining a region-of-interest (ROI) within the vitreous that contained all visible enhancement on the final image. The same ROI was then applied to all images in the set. The PS' of each lesion was calculated for all postinjection images based on the time between the center of Gd-DTPA injection and the center of image acquisition (approximately 5, 15 and 25 min for the first, second and third post-injection images, respectively). To reduce the potential for error, values for each post-injection image were used in the final calculation of PS' only if the amount of enhancement over the selected ROI reached a mean signal intensity that was 30% greater than the signal intensity on the control image (Tofts and Berkowitz, 1993), When more than one image met this criterion, the PS' values were averaged to obtain a single value for the ROI, Computation of PS' also requires a measure of the lesion size. This was accomplished as follows: spatial calibration was first applied using the known (10 cm) field of view. A line was then drawn free-hand along the length of leaky retina. This length, combined with the known slice thickness, was used to calculate the surface area of leakage for determination of PS'. The length of the entire retina was measured by drawing a line along the retinal surface between the apparent locations of the ciliary body profiles. Since focal (superior and inferior) lesions were observed in most eyes that were studied, the size of the lesion was quantitatively expressed as a percentage of half the
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total retinal length. Both measurements were obtained from the earliest post-contrast injection image. The earliest post-contrast image was selected for measurement of lesion size because the leakage could be identified, yet the effect of contrast diffusion into the vitreous on the interpretation of lesion size was minimal compared to the later images. Analysis of focal lesions was straightforward. However, in some eyes, diffuse BRB lesions covered most or all of the retinal surface. Diffuse lesions were analysed in two ways: first, the lesion was considered as a whole and an ROI was placed around the entire area of leakage; and second, separate ROIs were drawn around areas of enhancement in the superior and inferior halves of the vitreous. The two ROIs intersected, but did not overlap, near the geometric center of the globe; all visible leakage was incorporated into a ROI. Separate analysis of superior and inferior leakage provided a means of investigating variability in PS' between the two regions. In addition, regional values could be compared to values obtained from focal lesion in the same anatomic locations. When the two methods of analysis were compared in eyes with diffuse leakage, no significant differences were found between the superior and inferior retinal PSi, and the PS' provided by measuring the entire leakage as a whole was similar to the PS' values provided by measuring each half. In the results, PS' values were given for the superior and inferior hemi-retina to provide consistency in data reporting between eyes with focal lesions and eyes with diffuse lesions.
Fluorescein Angiography Fluorescein angiography and histology (see methods below) were performed to corroborate and extend the MRI findings. On post-ischemia day 58, fundus photography and fluorescein angiography were performed on eyes subjected to 60 or 100 min ofischemia (see Results). After administration of 10% sodium fluorescein (0.2 ml) via the marginal ear vein, fluorescein angiography was performed using a Topcon TRC-50X fundus camera (Topcon, East Paramus, N], U.S.A.).
Histology Eyes were obtained from animals that underwent 6 0 r a i n ( n = 3 eyes) or lOOmin ( n = 3 eyes) of ischemia and 58 days of reperfusion. In each eye, an incision was placed slightly posterior to the limbus and the specimens were immediately fixed in 2.5% paraformaldehyde and 2% glutaraldehydc at 4°C. After several weeks of fixation, the antero-posterior diameters of the eyes were measured for later comparison with the MRI measurement of ocular diameter. The mean diameters ( 1 7 . 0 m m by gross measurement and 17' 1 mm by MRI) were essentially the same, indicating that little tissue shrinkage had
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occurred during the process of fixation. This suggested that true lesion measurements could be obtained from fixed ocular specimens. In dissecting the globes, distinct regions of RPE clumping were observed. In eyes subjected to 60 min of ischemia, these regions had sharp, slightly irregular borders outside of which was grossly normal pigmentation. Gross histologic measurements were made to correlate with the size of retinal lesions observed using MRI. The vertical dimension of these lesions was measured at the posterior pole using a Castroviejo caliper. RPE clumping was uniform and diffuse in eyes that had undergone 100 min of ischemia. Therefore, measurement of lesion size was not necessary in these eyes. In eyes that underwent 60 min of ischemia, tissue samples were then obtained from within the center of the RPE disturbance and from the border between the pigmentary disturbance and clinically normal retina. The specimens were pre-embedded in agarose gel, processed by serial dehydration in increasing concentrations of alcohol and embedded in glycolmethacrylate. Sections (2.5/zm thick) were cut, mounted on glass slides and stained with double Lee's stain. Tissue was examined by light microscopy.
Statistical Analyses Where appropriate, comparisons were made between samples using the two-tailed Student's t-test. However, the relationship between time after ischemia or duration of ischemia and BRB parameters required statistical methods that accounted for the mixture of paired and unpaired data. This objective was accomplished by computing a slope for each animal, then determining if the m e a n slope differed significantly from zero. Since the data were not normally distributed, the WiIcoxon signed rank test was used to determine if the slope was significantly different from zero.
3. Results
BRB Lesion Size and Location The size and location of retinal leakage was dependent on the duration of ischemia and the time of reperfusion. Leakage was confined to the area over the medullary ray (inner BRB breakdown) 1 day after 20 min of ischemia (Fig. 1). Durations of ischemia of 40 min or longer usually produced some degree of outer BRB involvement. Most eyes exposed to 40 and 60 min of ischemia demonstrated outer BRB sparing in the posterior pole region (Table I). The leakage in eyes that underwent 80 or 100 min of ischemia was more diffusely distributed in the outer BRB. After 57 days of reperfusion, the location of persistent leakage in eyes exposed to 60 min of ischemia lay within the
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confines of the areas of leakage seen on post-ischemia day 1 (Fig. 2). Lesion size in eyes that demonstrated leakage on post-ischemia day 1 increased from 9 " 7 + 3 - 3 % ( m e a n _ S.E.M. ; n = 4) of the hemiretinal length after 2 0 m i n of ischemia to 100___0-0% ( n = 4 ) after 100 min of ischemia (Fig. 3). Superior loci of leakage accounted for all visible BRB involvement in the 20min ischemia group. By contrast, after 40 min of ischemia, the m e a n percentages of superior and inferior involvement were virtually identical: 4 1 " 4 + 1 7 " 2 % (n = 4 eyes) and 4 0 " 7 + 1 3 " 6 % (n = 4 eyes), respectively. After 60 min of ischemia, inferior lesions were, on average, larger t h a n superior lesions. When the data was pooled with post-ischemia day 1 data from animals that underwent 60 rain of ischemia for longitudinal studies (see below), the difference in lesion size became statistically significant (P = 0.005). Figure 4 shows the percent BRB involvement in eyes that were subjected to 60 min of ischemia for the purpose of longitudinal MRI studies. A statistically significant difference (P = 0-024) was observed on post-ischemia day 1 between the percent of superior (44"2 _+ 10"3 %, mean_+ S.E.M. ; n = 7 eyes) and inferior (70.5_+12.0%) retinal involvement. These values were essentially unchanged (45.9_+9.6% and 72"0 _+ 8.2 %; n = 11 eyes), but the level of significance increased (P = 0"005), by the addition of eyes from the time-response study above that had undergone 60 min of ischemia. Pooling is valid in this case because all eyes were imaged on the same day of reperfusion. This was not possible for other durations of ischemia because the longitudinal study examined only 60-min ischemic eyes. In the longitudinal study, both superior and inferior lesions decreased in size over time (Fig. 4). The m e a n slope of change in lesion size was statistically significant only for superior BRB defects (P = 0.031, Wilcoxon signed rank test). The m e a n difference in superior lesion sizes on days 1 and 57 was 40.6% of the hemiretinal length. Inferior lesions healed more slowly, and lesion sizes did not differ significantly on any 2 days during the time course. The m e a n difference in inferior lesion sizes on days 1 and 57 was 48"9% of the hemi-retinal length. The m e a n hemiretinal involvement on post-ischemia day 57 was 1 3 " 9 + 7 " 0 % ( n = 3 eyes) and 30"2_+15"1% ( n = 3 eyes) for superior and inferior lesions, respectively. However, because of the small n u m b e r of eyes examined and the absence of leakage in one eye, neither of these values was significantly greater t h a n zero.
BRB Permeability Studies Figure 5 shows the effect of O, 20, 40, 60, 80 and 100 min of ischemia on blood-retinal barrier permeability, normalized to the area of retinal leakage (PS') on day 1 after the ischemic insult. Eyes subjected
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FIG. 1. Representative images acquired 1 day after 20- to 100-min duration of ischemia. All images were taken approximately 25 min after Gd-DTPA injection. Inner BRB breakdown in the eye that underwent 20 min of ischemia (arrowhead) is indicated by a focal area of vitreous enhancement directly overlying the region of the vascularized medullary ray. Outer BRB breakdown first appears after 40 min of ischemia, as evidenced by leakage from the inferior (avascular) retina (lower arrowhead). With 40, 60 and 80 min of ischemia, an area of BRB sparing is found between superior and inferior loci of leakage (these foci are indicated by the two arrowheads on the 40 min ischemia image). Finally, in eyes subjected to 100 min of ischemia, no area of retina is spared from leakage.
TABLE I
Vertical dimensions of posterior retinal sparing in eyes exposed to ischemia and 1 day of reperfusion
Duration of ischemia*
Central retinal sparing (mm)l"
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40 60 80
7"7,-2-0"7 7"2 _-2-1-0 6-5+0"1
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* Includes only the durations of ischemia that produced isolated superior and inferior retinal leakage. I" Mean+s.E.M. No significant differences were noted between any two groups. :~ Eyes that developed diffuse leakage or eyes that showed no inferior BRB leakage were excluded.
to 0 a n d 20 m i n of ischemia provided m e a n PS' values that were n o t significantly greater t h a n zero, However, between 2 0 m i n a n d 1 0 0 rain of ischemia, m e a n PS'
of both superior a n d inferior lesions increased with the time of ischemia in a n e a r l y linear fashion. The m e a n slope of c h a n g e in PS' was significantly different from zero for both superior (P = 0"002, Wilcoxon signed r a n k test) a n d inferior (P = 0-017) lesions. The greatest values of PS' were observed in eyes subjected to 100 m i n of ischemia, which averaged 5-03__+0"95 x 10 4 cm m i n -1 (mean+s.E.M., n = 4 eyes) for superior lesions a n d 5 . 1 9 + 0 ' 4 2 x 1 0 4 (n = 4 eyes) for inferior lesions. No statistically significant difference was observed between the permeabilities of superior a n d inferior lesions at a n y d u r a t i o n of ischemia. The time-course of PS' in eyes subjected to 60 rain of isehemia is presented in Figure 6. Note that on day 1 following ischemia, the m e a n PS' values obtained for lesions in these eyes ( 2 - 1 0 x l O - 4 c m m i n -1 a n d 20.9 x 10 4 cm rain -~ for superior and inferior lesions, respectively: n = 7 eyes) were consistent with PS' values obtained from other eyes that were treated the same (2.09 x 10 -4 cm m i n -1 a n d 2.28 x 10-1; n = 4
FIG. 2. Representative images of an eye that was exposed to 60 min of ischemia and then imaged longitudinally at the various post-ischemia times listed. As in Fig. 1, these images were taken at approximately 25 min after Gd-DTPA injection. Note that the leakage, while it does decrease in severity with time, does not completely disappear from this eye, even after 57 days of recovery (arrowheads). As with the 60-min ischemic eye shown in Fig. 1, the central area of retina is spared from BRB breakdown.
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Duration ofischemia (min) Fro. 3. Percentage hemiretinal involvement (mean _ S.~.M.) with leakages as a function of duration of ischemia. Measurements were from images obtained 1 day after the ischemic event. Numbers were computed by dividing the length of retinal leakage in the superior or inferior portions of the eye by half the total retinal length ( x 100%). Each point represents the a~erage of four eyes. There were no significant differences between superior ([~) and inferior (O) lesion sizes at any duration of ischemia, although there was a trend toward larger inferior lesions at 60 and 80 min of ischemia (see text and Fig. 4). The relationship between time of ischemia and lesion size, however, was statistically significant (P = 0'007 and P = 0'008, Wilcoxon signed rank test for all slopes).
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Fro. 5. Permeability-surface-area product, normalized per unit area of retinal leakage (mean PS'_+ S.E.M.), as a function of the duration of ischemia. All data was obtained from images acquired on post-ischemia day 1. The increase in PS' between 20 min and 100 min of ischemia was nearly linear. Linear regression yielded fits of - 8 " 4 7 x 1 0 - 5 + 5 - 6 5 x lO-~(x) for the superior lesions ( & = O - 6 9 ) and --1"35 x 10-4+ 5"96 x lO-~(x) (r ~ = 0-78) for inferior lesions. There was no significant difference between superior ([-]) and inferior ((3) lesion PS' at any duration of ischemia. The slope of change in PS' for all animals was significantly different from zero for both superior (P = 0'002) and inferior (P = 0.027) lesions (Wilcoxon signed rank test). All values represent the means of four eyes.
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FIG. 4. Percentage hemiretinal leakage (mean__+S.E.M.) as a function of time after ischemia in eyes exposed to 60 min of ischemia and then imaged on days 1 (seven eyes), 3 (six eyes), 8 (six eyes), 15 (three eyes), 29 (three eyes) and 57 (three eyes). The difference between superior ([-]) and inferior {©) lesion sizes is statistically significant (P ~< 0.05, t-test) on days 1 and 8. When post-ischemia day 1 eyes are pooled with 60-min ischemia eyes from Fig. 3, the difference in lesion size is maintained. The decrease in the length of leaky retina over time was statistically significant for superior lesions only (P-- 0"03, Wilcoxon signed rank test for all slopes between days 1 and 57).
eyes) (Fig. 5). M e a n PS' decreased w i t h time after ischemia, b u t the m a j o r i t y of c h a n g e o c c u r r e d in t h e first 8 d a y s of t h e time course. However, due to t h e low n u m b e r of a n i m a l s in this sample, t h e slope of c h a n g e in PS' over the entire time c o u r s e w a s n o t statistically significant by n o n - p a r a m e t r i c analysis for the inferior lesions a n d of borderline significance for s u p e r i o r lesions (P = 0-06). The m e a n PS' values w e r e n o t
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FIG. 6. Permeability-surface-area product, normalized per unit area of retinal leakage (mean PS'_+ S.E.M.), as a function of time after a 60 min interval of ischemia. There is no significant difference in PS' between superior (D) and inferior (©) lesions throughout the time course. The slope of change in PS' between days 1 and 5 7 was different from zero at a significance level of P = 0.06 for superior lesions and P = 0.56 for inferior lesions (Wilcoxon signed rank test). The number of eyes per time point is described in Fig. 4.
significantly different from zero after p o s t - i s c h e m i a d a y 3 (superior lesions) or d a y 15 (inferior lesions).
Fluorescein Angiographic Findings The a p p e a r a n c e of r e t i n a l lesions p r o d u c e d by 60 a n d 1 0 0 m i n of i s c h e m i a w a s d o c u m e n t e d by f u n d u s p h o t o g r a p h y a n d fluorescein a n g i o g r a p h y in selected a n i m a l s at a p p r o x i m a t e l y 2 m o n t h s post-ischemia. These studies confirmed a d i s t r i b u t i o n of r e t i n a l d a m a g e t h a t c o r r e s p o n d e d to a r e a s of observed l e a k a g e o n c o n t r a s t - e n h a n c e d MRI. One m o n t h after 60 m i n
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Fro. 7. To better interpret the MP,I results, clinical photographs and fluorescein angiograms were performed on selected eyes that underwent ischemia and 58 days of reperfusion. (A) Fundus photograph of 58-day-old inferior retinal lesion in eye exposed to 60 min of ischemia. Note the sharply demarcated area of pigmentary clumping and depigmentation. The area of retinal superior to the lesion appears clinically normal which is in agreement with the MRI finding of BRB sparing in the posterior pole of these eyes. (B) Late-phase fiuorescein angiogram of the same lesion shown in (A), shows hyperfluorescence consistent With a window-type defect. Leakage is assumed to be present, but is not as clearly seen as on the MR images obtained 1 day earlier. (C) Late-phase fluorescein angiogram of superior region of the same eye shown in (A) and (B). Note the pigmentary change just superior to the medullary ray which suggests that the retinal pigment epithelium (outer BRB) had been damaged in this location. Therefore, it is likely that superior leakage in this eye was due, at least in part, to outer BRB damage. (D) Fluorescein angiogram of another eye that was subjected to 60 min of ischemia. The superior border of this large 58-day-old lesion could not be photographed because of its peripheral location. This illustrates the variability in the clinical impression of lesion size in eyes subjected to the same duration of ischemia, a finding which is reflected by the variability in lesion size on the MR images (Figs 3 and 4). of retinal ischemia, geographic areas containing retinal pigment epithelial clumping and depigmentation were found (Fig. 7). These areas were sharply demarcated and had slightly irregular borders. They were confined to the inferior retina and an area around, but primarily superior to the medullary ray. On fluorescein angiography, transmission defects were observed that corresponded to the areas of pigmentary loss, but leakage (as defined by spread of fluorescence beyond the borders of these well-defined lesions) was not seen. Eyes exposed to 1 O0 min of ischemia differed only in the extent of pigmentary disturbance, which was uniformly distributed throughout the fundus.
Gross and Histopathologic Studies To determine whether a relationship exists between early disruption of the BRB and the eventual size of the macroscopic pigmentary lesion, the lengths of leaky
retina for each lesion on post-ischemia day 1 were compared to the gross vertical measurements of the corresponding pigmentary lesions in 3 eyes exposed to 60 min of ischemia followed by 58 days of reperfusion (Table II). In general, the vertical length of leakage was greater than the size of the pigmentary lesion. This difference was statistically significant for the inferior lesions (P = 0.04, t-test). Chronic pigmentary lesion size was significantly correlated with the size of the early BRB defect (P = 0.019, r 2 = 0.78, ANOVA). Pigmentary lesions from eyes that were exposed to 1 O0 min of ischemia were not measured because they were uniformly distributed throughout the retina as was the retinal leakage in these same eyes. Light microscopy was conducted on specimens from all three of the eyes that had undergone 60 min of retinal ischemia and 58 days of reperfusion. The lesions showed a marked derangement of the RPE (hypertrophy and hyperplasia) with varying degrees of
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FIG. 8. (A) Light micrograph taken of the border of an inferior area of pigmentary change as illustrated in Fig. 7(A) and (B). This eye had been subjected to 60 min of ischemia followed by 58 days of reperfusion. Note hypertrophy and hyperplasia of the RPE in the lesion (left of arrow) and the relatively normal appearance over an area which corresponded to clinically normal retina (right of arrow). (B) Area from same eye showing severe atrophic changes in the neural retina within a region of RPE injury. Pigment migration into the outer retinal layers is also observed (arrows) (Lee's stain: x 250). atrophy of the overlying retina (Fig. 8). However, areas that were devoid of RPE cells were not found. In some lesions, pigment had migrated into the outer neural retina (Fig. 8). Superior lesions showed similar qualitative changes as did inferior lesions. In most sections, RPE damage appeared patchy. Therefore, it was difficult to determine whether all RPE abnormalities lay within the confines of the clinical lesions that were observed. 4. Discussion In this study, contrast-enhanced MRI was used to localize and quantitate BRB breakdown after pressureinduced ischemia in the rabbit eye. Localization of the leakage provided a means of identifying its source: inferior retinal leakage could be attributed entirely to lesions at the level of the RPE (outer BRB) since this region does not possess retinal blood vessels and the only other source of Gd-DTPA is from the choroidal vasculature. Superior lesions could represent either outer or inner BRB breakdown. However, the likelihood of outer BRB barrier breakdown in this location could sometimes be inferred from the size of the lesion,
such as when lesions were clearly larger t h a n the vertical diameter of the medullary ray. Several types of quantitative physiologic information could be extracted from the images: (1) the n u m b e r of macroscopic BRB lesions, (2) the size of the lesions along the length of retina in the selected MRI slice, (3) the permeability of the lesions normalized per unit area of leaky retina (PS') and (4) the time-course of changes in these parameters. PS' and lesion size measured on post-ischemia day 1 increased with duration of ischemia. The PS' reached a mean of 5.2 x 10 -4 cm min -1 with a 100% extent of retinal involvement in eyes subjected to 100 min of ischemia. It is doubtful that durations of ischemia longer than 100 min would have resulted in further increases in PS'. This contention is supported by previous studies in which contrast-enhanced MRI was used to quantitate the permeability of RPE lesions induced by intravenous sodium iodate. Sodium iodate caused a severe, widespread disruption of the outer BRB and resulted in a m e a n PS' value of 5.65 + 0 . 2 5 × 10 4 cm min 1 (n = 6 eyes) (Berkowitz et al., 1992). This value is not significantly different than the PS' produced by 100 min of ischemia, but it is greater (P ~< 0.05, t-test) than that produced by 80 min of ischemia. Although BRB lesions 'healed' over time, this process appeared very slow. Therefore, overall changes in inferior lesion size and permeability were not statistically significant at our sample size. Superior lesions showed a decrease in permeability that was of borderline statistical significance (P = 0.06). At 57 days, leakage was still observed in two of three eyes examined. Histologic sections demonstrated hypertrophy and hyperplasia of RPE cells, but the RPE formed a continuous layer. Therefore, restoration of BRB function appears to require longer than the time required to simply repopulate the region with viable RPE cells. One of three eyes that underwent MRI on post-ischemia day 57 showed no leakage, but it is not known whether all eyes exposed to 60 min ofischemia would ultimately show a complete restoration of BRB function with respect to the Gd-DTPA tracer. The focal distribution of BRB breakdown in eyes exposed to intermediate durations of ischemia (40 to 80 min) suggested that certain areas of the RPE were resistant to ischemic injury. One obvious area of RPE 'sparing' was found in avascular retina immediately beneath the medullary ray. Marmot and Dalal recently studied histologic, ultrastructural and electrophysiologic aspects of the pressure-ischemic rabbit eye and found that ischemic damage to the neural retina did not occur in a uniform fashion (Marmot and Dalal, 1993). Instead, they found patchy areas of tissue injury in the neural retina. With regards to the RPE, a transition zone was described between an area of preservation and disruption in an eye exposed to 60 min of ischemia; however, they did not describe the location of this zone. Hughes found an anatomic
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TABLE II
Vertical dimensions of grossly visible retinal lesions produced by 60 rain of retinal ischemia and 58 dabs of reperfusion compared to size of retinal lesions on images acquired after 1 day of reperfusion Superior lesions Eye 1
2 3 mean + S.E.M.
MRI measurement of lesion size* 5-0 4:'3 12"0 7'1 + 2"6
Inferior lesions
Gross measurement of lesion size 4"0 5"3 7"5 5"6 + 1'0
MRI measurement of lesion size 10"4 9"5 10"8 10"2 + 0"4t
Gross measurement of lesion size 7-2 8'0 8'0
7"7 +_0"31-
* All measurements in millimeters. t P = 0-04, t-test. susceptibility of the RPE to pressure-induced ischemia in the rat eye (Hughes, 1991). In eyes that underwent 2 hr of ischemia and 2 weeks of reperfusion, peripheral areas of RPE remained intact while central areas showed extreme attenuation or absence of the RPE. A transition zone was also identified that showed metaplastic changes in the RPE cells. The results of the present study suggest that a different pattern of ischemic damage occurs in the rabbit RPE. Using leakage as an indicator of RPE damage, the most susceptible areas of ischemia were found in the inferior periphery and immediately superior to the medullary ray. This pattern of damage was confirmed by clinical examination as well as, to some extent, by light microscopy. It is not clear as to why certain areas of the outer BRB appear more susceptible to damage t h a n others. One possibility is that the RPE is less able to withstand a given duration of ischemia in certain areas, perhaps because of lower stores of energy substrates or greater accumulation of toxic metabolic by-products. Another explanation concerns the reperfusion period. In various tissues, ischemia-reperfusion results in damage during the reperfusion phase as well as in the ischemic period (McMillen, Huribal and Sumpio, 1993). Reperfusion injury has been attributed to a no-reflow phenomenon resulting from microvascular occlusion by leukocytes. Therefore, it is possible that selected areas of the rabbit choroidal vasculature are more susceptible to the no-reflow p h e n o m e n o n than others. Another aspect of BRB damage in this study concerns its possible relationship to loss of neuroretinal function. Although electroretinographic (ERG) measurements were not done in this study, it is possible to compare the extent of BRB damage (size of the lesion) with the loss of ERG wave amplitudes observed by other investigators in the pressureischemic rabbit eye. The work of Ulrich and Reimann (1986) proved useful in this regard. Their study focused on the b-wave amplitude because it is the most sensitive component of the ERG to ischemia (Foulds and Johnson, 1974). The b-wave amplitudes recorded one day after complete ischemia in their study was
100%, 80%, 2 5 % and 0% of normal after ischemic intervals of 30, 40, 60 and 90 min, respectively. After 20 min of ischemia, 95 % sparing was observed, which is somewhat consistent with the 100% recovery of b-wave amplitude after 30 min of ischemia. After 100 min of ischemia, there was 0 % BRB sparing, which compares reasonably well with the 0 % recovery of b-wave amplitude that was observed following 90 min of ischemia. Therefore, general agreement is observed between the size of BRB lesions and ERG bwave amplitudes. However, quantitative differences should be expected between these endpoints because ERG and contrast-enhanced MR/ provide different types of functional information. In this study, it was not possible to measure inner BRB breakdown with certainty, although the superior leakage found in m a n y eyes was strongly suggestive of its occurrence. In eyes exposed to 20 min of ischemia, focal leakage was seen in several eyes that remained isolated to the area overlying the medullary ray. In one eye exposed to 40 min of ischemia, superior leakage was comprised of two juxtaposed areas of leakage: one very focal area over the medullary ray and another larger area just superior to the first (image not shown). Because the leakage from these areas blended together somewhat on all images, it was analyzed as a single lesion. However, on the basis of location and focality of the leakage, we attributed the first of the two areas of leakage to inner BRB breakdown. Lesion size and distribution identified by M R / o n day 1 after retinal ischemia correlated significantly with measurements of pigmentary lesions obtained from gross ocular specimens 2 months after the ischemia (Table n). However, the m e a n lesion size predicted by M R / w a s larger than the final RPE lesion size. There are two possible explanations for this observation: first, leakage measured on the images was, in fact, larger t h a n the actual area of increased permeability. This might occur when, during the elapsed time of image acquisition, some Gd-DTPA diffuses laterally from the edges of the lesion. In order to minimize this error, lesion sizes were measured from the first post-
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injection image. Second, the increased area of permeability m a y have included a ' w a t e r s h e d ' area of injured RPE that initially leaked, but was able to retain a normal clinical appearance during the 2-month reperfusion period. We suspect that a combination of these possibilities may have accounted for the overestimation of RPE lesion size. Although quantitative information regarding BRB permeability is not available in this model with which to compare these results, morphologic studies do lend support to some of the findings. Minimal leakage was observed in eyes subjected to 20 min of ischemia, but there was no inferior leakage in these eyes to suggest outer BRB breakdown had occurred. This is consistent with the detailed histologic and ultrastructural studies of Johnson and Foulds who investigated the effects of pressure-ischemia on the retina of the Dutch-belted rabbit (Johnson and Foulds, 1978). They found that an ischemic interval of 15 min produced no acute injury to the RPE. However, 30 min of ischemia or longer caused vacuolization of the RPE cell cytoplasm. More severe changes, which included cellular distortion, were observed after 90 and 1 2 0 m in of ischemia. They also observed microscopic edema of the nerve fiber layer in the medullary ray and vacuolization of endothelial cells in the preretinal blood vessels after only 15 min of ischemia. This lends support to the contention that inner BRB breakdown accounted for the mild leakage observed at the location of the medullary ray after only 20 min of ischemia in the present study. The effect of total ischemia on the BRB could be modulated by a variety of factors, including energy substrate availability, local retinal temperature, state of retinal dark adaptation, and choice and dose of anaesthetics [ketamine has been found to exert a protective effect on the neural retina during ischemia, but its effect on the RPE is not k n o w n (Tsukahara et al., 1992)]. In addition, some authorities have suggested the possibility that mechanical factors related to elevated intraocular pressure could account for some of the tissue damage encountered in this model (Blair et al., 1989; Marmor and Dalai, 1993). However, the absence of any direct evidence of mechanical damage has cast doubt on this explanation (Marmor and Dalal, 1993). An effort to thoroughly address the influence of various modulating factors on injury to the BRB in this model is currently underway in our laboratory. In summary, several biologic questions regarding BRB function in the pressure-ischemic rabbit eye were addressed in this study. First, BRB breakdown is not a uniform phenomenon, but rather a ' p a t c h y ' process involving several predisposed areas. This is in agreement with previous morphologic evidence (Marmor and Dalal, 1993). It becomes more uniform at ischemia durations of 80 min or longer. Secondly, the threshold for BRB damage occurs between 20 and 40 min duration of ischemia. Thereafter, increasing
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the duration of ischemia produces a graded increase in BRB permeability through l O O m i n of ischemia. Finally, BRB breakdown decreases with time after ischemia, but repair is a long process and complete restoration of function m a y not always occur. The relevance of these findings to h u m a n retinal disease remains to be determined. Interruption of blood flow to both the retina and choroid could occur in some instances such as acute glaucoma or ophthalmic artery occlusion, but it is not typical of most ischemic retinopathies in humans. In addition, there are differences in the enzymology of rabbit and h u m a n retinas, particularly with regards to energy snbstrate metabolism (Lowry et al., 1961; Lowry, Roberts and Lewis, 1956; Ames and Li, 1992). Such differences must be considered before extrapolating these results to other species. However, investigations of variables that might influence the susceptibility of the BRB to ischemic injury in the rabbit eye could lead to new treatment strategies, which then could be examined in other animal models.
Acknowledgements Supported by a grant from the Juvenile Diabetes Foundation, Int., New York (Dr Wilson) and an unrestricted grant from Research to Prevent Blindness (RPB), New York. Additional support provided by the National Center for Research Resources and the Biomedical Research Technology Program (BRTP). Dr Berkowitz is the recipient of a RPB career development award. The authors thank ]ackye Lambert for her expert technical assistance. Statistical consultation and analyses were provided by Dr Tom Carmody.
References Ames, A. and Li, Y.Y. (1992). Energy requirements of glutamatergic pathways in rabbit retina. J. Neurosci. 12, 4234-42. Berkowitz, B. A., Sato, Y., Wilson, C. A. and de Juan, E., Jr (1991). Blood-retinal barrier breakdown investigated by real-time magnetic resonance imaging after gadolinium-diethylenetriaminepentaacetic acid injection. Invest. Ophthalmol. Vis. Sci. 32, 2854-2860. Berkowitz, B. A., Torts, P. S., Sen, H. A., Ando, N. and de Juan, E., Jr (1992). Accurate and precise measurement of blood-retinal barrier breakdown using dynamic GdDTPA MRI. Invest. Ophthalmol. Vis. Sci. 33, 3500-6. Blair, N.P., Baker, D.S., Rhode,J. P. and Solomon, M. (1989). Vitreoperfusion: a new approach to ocular ischemia. Arch Ophthalmol 107, 417-23. Cunha-Vaz, J. G. (1979). The blood-ocular barriers. Surv. Ophthalmol. 23, 279-96. Ferris, F.L. and Patz, A. (1984). Macular edema. A complication of diabetic retinopathy. Surv. Ophthalmol. 28, 452-61. Foulds, W.S. and Johnson, N.F. (1974). Rabbit electroretinogram during recovery from induced ischemia. Trans. Ophthalmol. Soc. UK 94, 383-93. Hayreh, S.S., Kolder, H.E. and Weingeist, T.A. (1980). Central retinal artery occlusion and retinal tolerance time. Ophthalmology 87, 75-8. Heike, M. and Marmor, M.F. (1991). Recovery of retinal pigment epithelial function after ischemia in the rabbit. Invest. Ophthalmol. Vis. Sci. 32, 73-7.
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Hughes, W. F. (1991). Quantitation of ischemic damage in the rat retina. Exp. Eye Res. 53, 573-82. Johnson, N. F. and Foulds, W. 8. (1978). The effects of total acute ischemia on the structure of the rabbit retina. Exp. Eye Res. 27, 45-59. Kaskel, D., Valenzuela, H., Hockwin, 0., Muntau, M. and Schedtler, C.-M. (1976). Enzymologic and histologic investigations in normal and pressure-ischemic retina of rabbits. Graefe's Arch. Clin. Exp. Ophthalmol. 200, 71-8. Lowry, O.H., Roberts, N.R. and Lewis, C. (1956). The quantitative histochemistry of the retina. ]. Biol. Chem. 220, 879-92. Lowry, O. H., Roberts, N. R., Schulz, D. W., Clow, J. E. and Clark, J.R. (1961). Quantitative histochemistry of retina. II. Enzymes of glucose metabolism. ]. Biol. Chem. 236, 2 8 t 3 - 2 0 . Marmor, M. F. and Dalai, R. (1993). Irregular retinal and RPE damage after pressure-induced ischemia in the rabbit. Invest. Ophthalmol. Vis. Sci. 34, 2570-5.
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McKechnie, N. M., Johnson, N. F. and Foulds, W. S. (1982). The combined effects of light and acute ischemia on the structure of the rabbit retina: a light and electron microscopic study. Invest. Ophthalmol. Vis. Sci. 22, 449-59. McMillen, M.A., Huribal, M. and Sumpio, B. (1993). Common pathway of endothelial-leukocyte interaction in shock, ischemia, and reperfusion. Am. ]. Surg. 166, 557-562. Tofts, P. S. and Berkowitz, B. A. (1993). Rapid measurement of capillary permeability using the early part of the dynamic Gd-DTPA MRI enhancement curve. ]. Magn. Res. 102, 129-36. Tsukahara, Y., Blair, N.P., Spellman Eappen, D. C., Moy, J.J., Takahashi, A., Shah, G.K. and Viana, M. A. G. (1992). Ketamine suppresses ischemic injury in the rabbit retina. Invest. Ophthalmol. Vis. Sci. 33, 1822-5. Ulrich, W.-D. and Reimann, J. (1986). Survival and revival times of the retina: ERG studies at total ocular ischemia in rabbits and cats. Doc. Ophthalmol. 63, 91-9.
Blood-Retinal Barrier Breakdown Following Experimental Retinal Ischemia and Reperfusion C H A R L E S A . W l L S O N a*, B R U C E A . BERKOWlTZa, b,L H I D E H A R U F U N A T S U ~.d, D A V I D C..M'ETRIKINL D A V I D W . H A R R I S O N " , M I C H A E L K . LAMa AND PETER L. S O N K I N " Departments of aOphthalmology, bRadiology, and cBiomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX, U.S.A. and ~Department of Ophthalmology, Tokyo Women's Medical College, Tokyo, Japan ,(Received Houston 25 January 1995 and accepted in revised form 6 June 1995) The purpose of this study was to examine the effects of acute ischemia and reperfusion on blood-retinal barrier (BRB) function in the rabbit eye. Hydrostatic pressure (140 mmHg) was used to create total retinal ischemia for intervals of 20, 40, 60, 80 or 100 min in the rabbit eye, The location, size and permeability-surface-area product normalized to the area of retinal leakage (PS') of ischemia-induced BRB lesions were then measured using contrast-enhanced magnetic resonance imaging (MRI) after various intervals of reperfusion. Diffuse outer BRB leakage occurred in most eyes subjected to 80 or 100 min of ischemia. A posterior region of outer BRB sparing was found in eyes that underwent lesser durations of ischemia. On day 1 after retinal ischemia, a linear relationship was found between mean PS' and duration of ischemia for periods of ischemia between 20 and 100 min [slope: 5.65 x 10 -6 to 5.96 × 10 -G cm min 1 (min ischemia)-l; r 2 ~> 0-69]. Lesion size also increased between 20 and 1013min of ischemia. In a longitudinal study, eyes exposed to 60 rain of ischemia showed a decrease in PS and a lesion size over an 8-week period of observation. However, leakage was still present on post-ischemia day 57 in two of three eyes examined. Data obtained in these experiments are expected to prove useful in future studies aimed at understanding how BRB damage relates to neuroretinal damage after ischemia-reperfusion injury. © 1995 Academic Press Limited Key words: blood-retinal barrier, ischemia-reperfusion, rabbit, magnetic resonance imaging.
1. Introduction Ischemia is a component of m a n y retinal and choroidal vascular diseases. While acute retinal ischemia can cause direct cellular injury or death (Foulds and Johnson, 1974: Marmor and Dalai, 1993; Kaskel et al., 1976; Johnson and Foulds, 1978), the relative contribution of post-ischemic sequelae, such as disruption of the blood-retinal barrier (BRB), to retinal damage is less certain. In theory, ischemia sufficient to cause severe injury to the retinal vascular endothelium (inner BRB) a n d / o r retinal pigment epithelium (outer BRB) would be expected to cause influx of bloodderived water and solutes (exudate) into the retina (Cunha-Vaz, 1979), ultimately leading to permanent structural damage (Ferris and Patz, 1984). In addition, migration of exudate outside the primary area of BRB injury could result in increased visual morbidity, such as often occurs in diabetic macular edema (Ferris and Patz, 1984). Knowledge of the effects of ischemia on retinal structure and function has been furthered by use of animal models. For experimental purposes, retinal and choroidal ischemia is commonly achieved by elevating
* For correspondence at: Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9057, U.S.A.
0014-4835/95/110547+ 11 $12.00/0
the intraocular pressure until ocular blood flow is stopped. The histologic and electroretinographic effects of this procedure on the retina have been described previously in several species (Heike and Marmor, 1991 ; Foulds and Johnson, 1974; Marmor and Dalai, 1993; McKechnie, Johnson and Foulds, 1982; Johnson and Foulds, 1978). However, the effect of pressure-ischemia on blood-retinal barrier integrity has not been investigated in detail. In this study, contrast-enhanced magnetic resonance imaging (MRI) was used to quantitate the effects of ischemia on BRB breakdown in the rabbit eye. As has been shown in previous studies (Berkowitz et al., 1991, 1992), the MRI procedure provides a means of localizing leakage as well as measuring BRB permeability and lesion size. Such studies are facilitated by available MRI contrast agents that are freely diffusable and similar to fluorescein dye in molecular weight. Several biologic questions are addressed in this study. The first set of questions extends from a previous report of the light microscopic findings in the pressureischemic rabbit eye (Marmor and Dalai, 1993). The report indicated that neuroretinal and retinal pigment epithelial damage had occurred in a non-uniform or ' p a t c h y ' fashion. From this information, two questions were posed: does pressure-ischemia damage the BRB uniformly or is the damage non-uniform as suggested © 1995 Academic Press Limited
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by the morphologic data ? and, if non-uniform, what portions of the BRB are most susceptible to ischemic injury ? Another issue in the rabbit model relates to tolerance to ischemia. Hayreh and colleagues observed a threshold duration of ischemia that would result in irreversible retinal damage in the pressure-ischemic primate eye (Hayreh, Kolder and Weingeist, 1980). Yet, electroretinographic studies in the rabbit and cat eye have suggested a graded response to increasing duration of ischemia (Foulds and Johnson, 1974; Ulrich and Reimann, 1986). This raises the following questions regarding the behavior of the BRB: does a graded relationship exist between duration of ischemia and the severity of BRB breakdown ? Secondly, is there a threshold duration of ischemia that separates complete BRB recovery from acute damage ? In addition to the above considerations, the issue of cellular proliferation raises yet other questions. Injured retinal vascular endothelial and retinal pigment epithelial cells may remain capable of proliferation and, presumably, restoration of function. Therefore, the present study sought to determine whether, to what degree, and how rapidly BRB function is restored after ischemic damage in the rabbit model. 2. Materials and Methods
Animals and Anaesthesia Nineteen Dutch-belted (pigmented) rabbits weighing 1.9 to 2.0 kg were used in these experiments. Studies were conducted in accordance with institutional guidelines. Anaesthesia was induced by intramuscular injection of ketamine HC1 (25 mg kg -1) and xylazine HC1 (5 mgkg -1) and was maintained by intravenous infusion of ketamine HCI ( 4 0 m g k g - l h r -1) and xylazine ( 4 m g k g lhr-1) administered through a marginal ear vein. Pupils were dilated prior to ocular procedures with tropicamide HC1 (1%) and phenylephrine HC1 (2.5 %) eye drops.
Production of Ischemia A 20-gauge intravenous infusion catheter was placed in the anterior chamber at the limbus and secured with a 6-0 silk purse-string suture. The catheter was connected via intravenous infusion tubing to a bag of lactated Ringer's solution. The amount of pressure elevation at the level of the eye was adjusted by varying the height of the bag. During ischemia, bag height was adjusted to create a pressure of 140 mmHg at eye level; the pressure was confirmed using a calibrated transducer. Although arterial blood pressures were not monitored in these animals during ischemia, previous and subsequent experiments under similar conditions have revealed that systolic arterial pressures (measured from the auricular artery) range from 80 mmHg to 110 mmHg. Indirect ophthalmo-
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scopy was performed at the start of ischemia and periodically thereafter to confirm the absence of blood flow in the retinal blood vessels at the optic disk and along the medullary rays. In one animal, 0.2 ml of 10% sodium fluorescein was injected intravenously immediately after induction of ischemia. Fluorescein ophthalmoscopy showed no dye appearance in the eye until the pressure was released. After a given period of ischemia (see experimental protocols below), the pressure was lowered by removal of the anterior chamber catheter. In all eyes, indirect ophthalmoscopy revealed that blood flow was restored to the preretinal vasculature following catheter removal. In the fluorescein-injected animal, dye rapidly filled the anterior chamber upon removal of the catheter indicating that the anterior segment was reperfused. Corneo-scleral wounds were sutured closed using 8-0 polyglactin. An antibiotic ointment containing polymyxin B sulfate (10000 U g 1) and bacitracin zinc (500 U g i) was applied to the cornea at the end of the operative procedure.
Experimental Protocols In the first experiment, eyes were subjected to varying durations (20, 40, 60, 80 or 100 min) of ischemia and were examined using contrast-enhanced MRI 1 day later. Control eyes did not undergo ischemia, In one control eye, an anterior chamber catheter was inserted and later removed to control for the effects of surgical trauma. Twelve animals were divided into three groups of four animals. Their eyes were used in the experiment as follows: group 1 underwent 20 rain and (fellow eye) 49 min of ischemia, group 2 underwent 60 min and (fellow eye) 80 rain of ischemia, and group 3 underwent 100 min of ischemia and (fellow eye) no ischemia. Groups 1 and 2 were killed by intravenous KC1 injection immediately following the imaging procedure. Three of the animals in group 3 were killed on postischemia day 58, and the eyes were salvaged for histologic examination (see histology below). The fourth animal in this group died of suspected complications of anesthesia. In a second experiment, seven rabbits were subjected to a 60-min period of ischemia in one eye. These animals were then examined using MRI on postischemia days 1, 3, 8, 15, 29 and 57. Three of the seven animals completed all days of imaging. The others died during the follow-up period of suspected complications of anesthesia. On post-ischemia day 58, surviving animals were killed and their eyes salvaged for histologic examination (see histology below).
Magnetic Resonance Imaging During MRI, each animal was anesthetized as described above and mechanically ventilated via an endotracheal tube. The auricular artery was cannu-
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luted from which the heart rate, blood pressure and arterial blood gases were monitored. Ventilatory settings were adjusted to maintain the arterial blood gases to within a physiologically normal range (pH 7"35-7"45; PO 2 8 0 - 1 2 0 mmHg; PCO 2 2 0 - 4 0 mmHg). A warming pad was used to maintain core body temperature. MRI was performed on a 0.5T Toshiba clinical system (Toshiba America MRI, South San Francisco, CA, U.S.A.) using a 10 cm surface coil). Axial T1weighted images (TR = 400 ms and TE = 15 ms) with a slice thickness of 3"5 m m were obtained through the geometric center of both globes. These slices were oriented perpendicular to the long axis of the vascularized medullary ray. Each image required 6.9 rain to acquire. A control image was obtained followed by a bolus intravenous injection of contrast (Gd-DTPA; 1"0 mmol kg-1). Three sequential postinjection images were then acquired.
Image Analysis Images were transferred to a Macintosh llci data station and analysed using the program Image (version 1.50; W. Rasband, National Institutes of Health). A user-defined subroutine facilitated calculation of the permeability-surface area product normalized to the surface area of leaky retina (PS') according to previously published methods (Berkowitz et al., 1992). This measurement involved defining a region-of-interest (ROI) within the vitreous that contained all visible enhancement on the final image. The same ROI was then applied to all images in the set. The PS' of each lesion was calculated for all postinjection images based on the time between the center of Gd-DTPA injection and the center of image acquisition (approximately 5, 15 and 25 min for the first, second and third post-injection images, respectively). To reduce the potential for error, values for each post-injection image were used in the final calculation of PS' only if the amount of enhancement over the selected ROI reached a mean signal intensity that was 30% greater than the signal intensity on the control image (Tofts and Berkowitz, 1993), When more than one image met this criterion, the PS' values were averaged to obtain a single value for the ROI, Computation of PS' also requires a measure of the lesion size. This was accomplished as follows: spatial calibration was first applied using the known (10 cm) field of view. A line was then drawn free-hand along the length of leaky retina. This length, combined with the known slice thickness, was used to calculate the surface area of leakage for determination of PS'. The length of the entire retina was measured by drawing a line along the retinal surface between the apparent locations of the ciliary body profiles. Since focal (superior and inferior) lesions were observed in most eyes that were studied, the size of the lesion was quantitatively expressed as a percentage of half the
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total retinal length. Both measurements were obtained from the earliest post-contrast injection image. The earliest post-contrast image was selected for measurement of lesion size because the leakage could be identified, yet the effect of contrast diffusion into the vitreous on the interpretation of lesion size was minimal compared to the later images. Analysis of focal lesions was straightforward. However, in some eyes, diffuse BRB lesions covered most or all of the retinal surface. Diffuse lesions were analysed in two ways: first, the lesion was considered as a whole and an ROI was placed around the entire area of leakage; and second, separate ROIs were drawn around areas of enhancement in the superior and inferior halves of the vitreous. The two ROIs intersected, but did not overlap, near the geometric center of the globe; all visible leakage was incorporated into a ROI. Separate analysis of superior and inferior leakage provided a means of investigating variability in PS' between the two regions. In addition, regional values could be compared to values obtained from focal lesion in the same anatomic locations. When the two methods of analysis were compared in eyes with diffuse leakage, no significant differences were found between the superior and inferior retinal PSi, and the PS' provided by measuring the entire leakage as a whole was similar to the PS' values provided by measuring each half. In the results, PS' values were given for the superior and inferior hemi-retina to provide consistency in data reporting between eyes with focal lesions and eyes with diffuse lesions.
Fluorescein Angiography Fluorescein angiography and histology (see methods below) were performed to corroborate and extend the MRI findings. On post-ischemia day 58, fundus photography and fluorescein angiography were performed on eyes subjected to 60 or 100 min ofischemia (see Results). After administration of 10% sodium fluorescein (0.2 ml) via the marginal ear vein, fluorescein angiography was performed using a Topcon TRC-50X fundus camera (Topcon, East Paramus, N], U.S.A.).
Histology Eyes were obtained from animals that underwent 6 0 r a i n ( n = 3 eyes) or lOOmin ( n = 3 eyes) of ischemia and 58 days of reperfusion. In each eye, an incision was placed slightly posterior to the limbus and the specimens were immediately fixed in 2.5% paraformaldehyde and 2% glutaraldehydc at 4°C. After several weeks of fixation, the antero-posterior diameters of the eyes were measured for later comparison with the MRI measurement of ocular diameter. The mean diameters ( 1 7 . 0 m m by gross measurement and 17' 1 mm by MRI) were essentially the same, indicating that little tissue shrinkage had
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occurred during the process of fixation. This suggested that true lesion measurements could be obtained from fixed ocular specimens. In dissecting the globes, distinct regions of RPE clumping were observed. In eyes subjected to 60 min of ischemia, these regions had sharp, slightly irregular borders outside of which was grossly normal pigmentation. Gross histologic measurements were made to correlate with the size of retinal lesions observed using MRI. The vertical dimension of these lesions was measured at the posterior pole using a Castroviejo caliper. RPE clumping was uniform and diffuse in eyes that had undergone 100 min of ischemia. Therefore, measurement of lesion size was not necessary in these eyes. In eyes that underwent 60 min of ischemia, tissue samples were then obtained from within the center of the RPE disturbance and from the border between the pigmentary disturbance and clinically normal retina. The specimens were pre-embedded in agarose gel, processed by serial dehydration in increasing concentrations of alcohol and embedded in glycolmethacrylate. Sections (2.5/zm thick) were cut, mounted on glass slides and stained with double Lee's stain. Tissue was examined by light microscopy.
Statistical Analyses Where appropriate, comparisons were made between samples using the two-tailed Student's t-test. However, the relationship between time after ischemia or duration of ischemia and BRB parameters required statistical methods that accounted for the mixture of paired and unpaired data. This objective was accomplished by computing a slope for each animal, then determining if the m e a n slope differed significantly from zero. Since the data were not normally distributed, the WiIcoxon signed rank test was used to determine if the slope was significantly different from zero.
3. Results
BRB Lesion Size and Location The size and location of retinal leakage was dependent on the duration of ischemia and the time of reperfusion. Leakage was confined to the area over the medullary ray (inner BRB breakdown) 1 day after 20 min of ischemia (Fig. 1). Durations of ischemia of 40 min or longer usually produced some degree of outer BRB involvement. Most eyes exposed to 40 and 60 min of ischemia demonstrated outer BRB sparing in the posterior pole region (Table I). The leakage in eyes that underwent 80 or 100 min of ischemia was more diffusely distributed in the outer BRB. After 57 days of reperfusion, the location of persistent leakage in eyes exposed to 60 min of ischemia lay within the
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ET AL.
confines of the areas of leakage seen on post-ischemia day 1 (Fig. 2). Lesion size in eyes that demonstrated leakage on post-ischemia day 1 increased from 9 " 7 + 3 - 3 % ( m e a n _ S.E.M. ; n = 4) of the hemiretinal length after 2 0 m i n of ischemia to 100___0-0% ( n = 4 ) after 100 min of ischemia (Fig. 3). Superior loci of leakage accounted for all visible BRB involvement in the 20min ischemia group. By contrast, after 40 min of ischemia, the m e a n percentages of superior and inferior involvement were virtually identical: 4 1 " 4 + 1 7 " 2 % (n = 4 eyes) and 4 0 " 7 + 1 3 " 6 % (n = 4 eyes), respectively. After 60 min of ischemia, inferior lesions were, on average, larger t h a n superior lesions. When the data was pooled with post-ischemia day 1 data from animals that underwent 60 rain of ischemia for longitudinal studies (see below), the difference in lesion size became statistically significant (P = 0.005). Figure 4 shows the percent BRB involvement in eyes that were subjected to 60 min of ischemia for the purpose of longitudinal MRI studies. A statistically significant difference (P = 0-024) was observed on post-ischemia day 1 between the percent of superior (44"2 _+ 10"3 %, mean_+ S.E.M. ; n = 7 eyes) and inferior (70.5_+12.0%) retinal involvement. These values were essentially unchanged (45.9_+9.6% and 72"0 _+ 8.2 %; n = 11 eyes), but the level of significance increased (P = 0"005), by the addition of eyes from the time-response study above that had undergone 60 min of ischemia. Pooling is valid in this case because all eyes were imaged on the same day of reperfusion. This was not possible for other durations of ischemia because the longitudinal study examined only 60-min ischemic eyes. In the longitudinal study, both superior and inferior lesions decreased in size over time (Fig. 4). The m e a n slope of change in lesion size was statistically significant only for superior BRB defects (P = 0.031, Wilcoxon signed rank test). The m e a n difference in superior lesion sizes on days 1 and 57 was 40.6% of the hemiretinal length. Inferior lesions healed more slowly, and lesion sizes did not differ significantly on any 2 days during the time course. The m e a n difference in inferior lesion sizes on days 1 and 57 was 48"9% of the hemi-retinal length. The m e a n hemiretinal involvement on post-ischemia day 57 was 1 3 " 9 + 7 " 0 % ( n = 3 eyes) and 30"2_+15"1% ( n = 3 eyes) for superior and inferior lesions, respectively. However, because of the small n u m b e r of eyes examined and the absence of leakage in one eye, neither of these values was significantly greater t h a n zero.
BRB Permeability Studies Figure 5 shows the effect of O, 20, 40, 60, 80 and 100 min of ischemia on blood-retinal barrier permeability, normalized to the area of retinal leakage (PS') on day 1 after the ischemic insult. Eyes subjected
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FIG. 1. Representative images acquired 1 day after 20- to 100-min duration of ischemia. All images were taken approximately 25 min after Gd-DTPA injection. Inner BRB breakdown in the eye that underwent 20 min of ischemia (arrowhead) is indicated by a focal area of vitreous enhancement directly overlying the region of the vascularized medullary ray. Outer BRB breakdown first appears after 40 min of ischemia, as evidenced by leakage from the inferior (avascular) retina (lower arrowhead). With 40, 60 and 80 min of ischemia, an area of BRB sparing is found between superior and inferior loci of leakage (these foci are indicated by the two arrowheads on the 40 min ischemia image). Finally, in eyes subjected to 100 min of ischemia, no area of retina is spared from leakage.
TABLE I
Vertical dimensions of posterior retinal sparing in eyes exposed to ischemia and 1 day of reperfusion
Duration of ischemia*
Central retinal sparing (mm)l"
Number of eyes:~
40 60 80
7"7,-2-0"7 7"2 _-2-1-0 6-5+0"1
3 3 2
* Includes only the durations of ischemia that produced isolated superior and inferior retinal leakage. I" Mean+s.E.M. No significant differences were noted between any two groups. :~ Eyes that developed diffuse leakage or eyes that showed no inferior BRB leakage were excluded.
to 0 a n d 20 m i n of ischemia provided m e a n PS' values that were n o t significantly greater t h a n zero, However, between 2 0 m i n a n d 1 0 0 rain of ischemia, m e a n PS'
of both superior a n d inferior lesions increased with the time of ischemia in a n e a r l y linear fashion. The m e a n slope of c h a n g e in PS' was significantly different from zero for both superior (P = 0"002, Wilcoxon signed r a n k test) a n d inferior (P = 0-017) lesions. The greatest values of PS' were observed in eyes subjected to 100 m i n of ischemia, which averaged 5-03__+0"95 x 10 4 cm m i n -1 (mean+s.E.M., n = 4 eyes) for superior lesions a n d 5 . 1 9 + 0 ' 4 2 x 1 0 4 (n = 4 eyes) for inferior lesions. No statistically significant difference was observed between the permeabilities of superior a n d inferior lesions at a n y d u r a t i o n of ischemia. The time-course of PS' in eyes subjected to 60 rain of isehemia is presented in Figure 6. Note that on day 1 following ischemia, the m e a n PS' values obtained for lesions in these eyes ( 2 - 1 0 x l O - 4 c m m i n -1 a n d 20.9 x 10 4 cm rain -~ for superior and inferior lesions, respectively: n = 7 eyes) were consistent with PS' values obtained from other eyes that were treated the same (2.09 x 10 -4 cm m i n -1 a n d 2.28 x 10-1; n = 4
FIG. 2. Representative images of an eye that was exposed to 60 min of ischemia and then imaged longitudinally at the various post-ischemia times listed. As in Fig. 1, these images were taken at approximately 25 min after Gd-DTPA injection. Note that the leakage, while it does decrease in severity with time, does not completely disappear from this eye, even after 57 days of recovery (arrowheads). As with the 60-min ischemic eye shown in Fig. 1, the central area of retina is spared from BRB breakdown.
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Duration ofischemia (min) Fro. 3. Percentage hemiretinal involvement (mean _ S.~.M.) with leakages as a function of duration of ischemia. Measurements were from images obtained 1 day after the ischemic event. Numbers were computed by dividing the length of retinal leakage in the superior or inferior portions of the eye by half the total retinal length ( x 100%). Each point represents the a~erage of four eyes. There were no significant differences between superior ([~) and inferior (O) lesion sizes at any duration of ischemia, although there was a trend toward larger inferior lesions at 60 and 80 min of ischemia (see text and Fig. 4). The relationship between time of ischemia and lesion size, however, was statistically significant (P = 0'007 and P = 0'008, Wilcoxon signed rank test for all slopes).
.... I,,,,I
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10 20 30 40 50 60 70 80 90 100 Duration of ischemia (min)
Fro. 5. Permeability-surface-area product, normalized per unit area of retinal leakage (mean PS'_+ S.E.M.), as a function of the duration of ischemia. All data was obtained from images acquired on post-ischemia day 1. The increase in PS' between 20 min and 100 min of ischemia was nearly linear. Linear regression yielded fits of - 8 " 4 7 x 1 0 - 5 + 5 - 6 5 x lO-~(x) for the superior lesions ( & = O - 6 9 ) and --1"35 x 10-4+ 5"96 x lO-~(x) (r ~ = 0-78) for inferior lesions. There was no significant difference between superior ([-]) and inferior ((3) lesion PS' at any duration of ischemia. The slope of change in PS' for all animals was significantly different from zero for both superior (P = 0'002) and inferior (P = 0.027) lesions (Wilcoxon signed rank test). All values represent the means of four eyes.
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FIG. 4. Percentage hemiretinal leakage (mean__+S.E.M.) as a function of time after ischemia in eyes exposed to 60 min of ischemia and then imaged on days 1 (seven eyes), 3 (six eyes), 8 (six eyes), 15 (three eyes), 29 (three eyes) and 57 (three eyes). The difference between superior ([-]) and inferior {©) lesion sizes is statistically significant (P ~< 0.05, t-test) on days 1 and 8. When post-ischemia day 1 eyes are pooled with 60-min ischemia eyes from Fig. 3, the difference in lesion size is maintained. The decrease in the length of leaky retina over time was statistically significant for superior lesions only (P-- 0"03, Wilcoxon signed rank test for all slopes between days 1 and 57).
eyes) (Fig. 5). M e a n PS' decreased w i t h time after ischemia, b u t the m a j o r i t y of c h a n g e o c c u r r e d in t h e first 8 d a y s of t h e time course. However, due to t h e low n u m b e r of a n i m a l s in this sample, t h e slope of c h a n g e in PS' over the entire time c o u r s e w a s n o t statistically significant by n o n - p a r a m e t r i c analysis for the inferior lesions a n d of borderline significance for s u p e r i o r lesions (P = 0-06). The m e a n PS' values w e r e n o t
,,,[,,,,[i,,,I,,,,I,,,,I,,,,
0
10
20 30 40 50 Time after ischemia (days)
60
FIG. 6. Permeability-surface-area product, normalized per unit area of retinal leakage (mean PS'_+ S.E.M.), as a function of time after a 60 min interval of ischemia. There is no significant difference in PS' between superior (D) and inferior (©) lesions throughout the time course. The slope of change in PS' between days 1 and 5 7 was different from zero at a significance level of P = 0.06 for superior lesions and P = 0.56 for inferior lesions (Wilcoxon signed rank test). The number of eyes per time point is described in Fig. 4.
significantly different from zero after p o s t - i s c h e m i a d a y 3 (superior lesions) or d a y 15 (inferior lesions).
Fluorescein Angiographic Findings The a p p e a r a n c e of r e t i n a l lesions p r o d u c e d by 60 a n d 1 0 0 m i n of i s c h e m i a w a s d o c u m e n t e d by f u n d u s p h o t o g r a p h y a n d fluorescein a n g i o g r a p h y in selected a n i m a l s at a p p r o x i m a t e l y 2 m o n t h s post-ischemia. These studies confirmed a d i s t r i b u t i o n of r e t i n a l d a m a g e t h a t c o r r e s p o n d e d to a r e a s of observed l e a k a g e o n c o n t r a s t - e n h a n c e d MRI. One m o n t h after 60 m i n
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Fro. 7. To better interpret the MP,I results, clinical photographs and fluorescein angiograms were performed on selected eyes that underwent ischemia and 58 days of reperfusion. (A) Fundus photograph of 58-day-old inferior retinal lesion in eye exposed to 60 min of ischemia. Note the sharply demarcated area of pigmentary clumping and depigmentation. The area of retinal superior to the lesion appears clinically normal which is in agreement with the MRI finding of BRB sparing in the posterior pole of these eyes. (B) Late-phase fiuorescein angiogram of the same lesion shown in (A), shows hyperfluorescence consistent With a window-type defect. Leakage is assumed to be present, but is not as clearly seen as on the MR images obtained 1 day earlier. (C) Late-phase fluorescein angiogram of superior region of the same eye shown in (A) and (B). Note the pigmentary change just superior to the medullary ray which suggests that the retinal pigment epithelium (outer BRB) had been damaged in this location. Therefore, it is likely that superior leakage in this eye was due, at least in part, to outer BRB damage. (D) Fluorescein angiogram of another eye that was subjected to 60 min of ischemia. The superior border of this large 58-day-old lesion could not be photographed because of its peripheral location. This illustrates the variability in the clinical impression of lesion size in eyes subjected to the same duration of ischemia, a finding which is reflected by the variability in lesion size on the MR images (Figs 3 and 4). of retinal ischemia, geographic areas containing retinal pigment epithelial clumping and depigmentation were found (Fig. 7). These areas were sharply demarcated and had slightly irregular borders. They were confined to the inferior retina and an area around, but primarily superior to the medullary ray. On fluorescein angiography, transmission defects were observed that corresponded to the areas of pigmentary loss, but leakage (as defined by spread of fluorescence beyond the borders of these well-defined lesions) was not seen. Eyes exposed to 1 O0 min of ischemia differed only in the extent of pigmentary disturbance, which was uniformly distributed throughout the fundus.
Gross and Histopathologic Studies To determine whether a relationship exists between early disruption of the BRB and the eventual size of the macroscopic pigmentary lesion, the lengths of leaky
retina for each lesion on post-ischemia day 1 were compared to the gross vertical measurements of the corresponding pigmentary lesions in 3 eyes exposed to 60 min of ischemia followed by 58 days of reperfusion (Table II). In general, the vertical length of leakage was greater than the size of the pigmentary lesion. This difference was statistically significant for the inferior lesions (P = 0.04, t-test). Chronic pigmentary lesion size was significantly correlated with the size of the early BRB defect (P = 0.019, r 2 = 0.78, ANOVA). Pigmentary lesions from eyes that were exposed to 1 O0 min of ischemia were not measured because they were uniformly distributed throughout the retina as was the retinal leakage in these same eyes. Light microscopy was conducted on specimens from all three of the eyes that had undergone 60 min of retinal ischemia and 58 days of reperfusion. The lesions showed a marked derangement of the RPE (hypertrophy and hyperplasia) with varying degrees of
554
C.A. W I L S O N ET AL.
A
B ~
~ ~i i~!i ~ ~ ~
~ ~!~iiiiii~i:!:!~i:i~:!~:!iiii~:iiiiii:i!!:ii%~ ~
~
FIG. 8. (A) Light micrograph taken of the border of an inferior area of pigmentary change as illustrated in Fig. 7(A) and (B). This eye had been subjected to 60 min of ischemia followed by 58 days of reperfusion. Note hypertrophy and hyperplasia of the RPE in the lesion (left of arrow) and the relatively normal appearance over an area which corresponded to clinically normal retina (right of arrow). (B) Area from same eye showing severe atrophic changes in the neural retina within a region of RPE injury. Pigment migration into the outer retinal layers is also observed (arrows) (Lee's stain: x 250). atrophy of the overlying retina (Fig. 8). However, areas that were devoid of RPE cells were not found. In some lesions, pigment had migrated into the outer neural retina (Fig. 8). Superior lesions showed similar qualitative changes as did inferior lesions. In most sections, RPE damage appeared patchy. Therefore, it was difficult to determine whether all RPE abnormalities lay within the confines of the clinical lesions that were observed. 4. Discussion In this study, contrast-enhanced MRI was used to localize and quantitate BRB breakdown after pressureinduced ischemia in the rabbit eye. Localization of the leakage provided a means of identifying its source: inferior retinal leakage could be attributed entirely to lesions at the level of the RPE (outer BRB) since this region does not possess retinal blood vessels and the only other source of Gd-DTPA is from the choroidal vasculature. Superior lesions could represent either outer or inner BRB breakdown. However, the likelihood of outer BRB barrier breakdown in this location could sometimes be inferred from the size of the lesion,
such as when lesions were clearly larger t h a n the vertical diameter of the medullary ray. Several types of quantitative physiologic information could be extracted from the images: (1) the n u m b e r of macroscopic BRB lesions, (2) the size of the lesions along the length of retina in the selected MRI slice, (3) the permeability of the lesions normalized per unit area of leaky retina (PS') and (4) the time-course of changes in these parameters. PS' and lesion size measured on post-ischemia day 1 increased with duration of ischemia. The PS' reached a mean of 5.2 x 10 -4 cm min -1 with a 100% extent of retinal involvement in eyes subjected to 100 min of ischemia. It is doubtful that durations of ischemia longer than 100 min would have resulted in further increases in PS'. This contention is supported by previous studies in which contrast-enhanced MRI was used to quantitate the permeability of RPE lesions induced by intravenous sodium iodate. Sodium iodate caused a severe, widespread disruption of the outer BRB and resulted in a m e a n PS' value of 5.65 + 0 . 2 5 × 10 4 cm min 1 (n = 6 eyes) (Berkowitz et al., 1992). This value is not significantly different than the PS' produced by 100 min of ischemia, but it is greater (P ~< 0.05, t-test) than that produced by 80 min of ischemia. Although BRB lesions 'healed' over time, this process appeared very slow. Therefore, overall changes in inferior lesion size and permeability were not statistically significant at our sample size. Superior lesions showed a decrease in permeability that was of borderline statistical significance (P = 0.06). At 57 days, leakage was still observed in two of three eyes examined. Histologic sections demonstrated hypertrophy and hyperplasia of RPE cells, but the RPE formed a continuous layer. Therefore, restoration of BRB function appears to require longer than the time required to simply repopulate the region with viable RPE cells. One of three eyes that underwent MRI on post-ischemia day 57 showed no leakage, but it is not known whether all eyes exposed to 60 min ofischemia would ultimately show a complete restoration of BRB function with respect to the Gd-DTPA tracer. The focal distribution of BRB breakdown in eyes exposed to intermediate durations of ischemia (40 to 80 min) suggested that certain areas of the RPE were resistant to ischemic injury. One obvious area of RPE 'sparing' was found in avascular retina immediately beneath the medullary ray. Marmot and Dalal recently studied histologic, ultrastructural and electrophysiologic aspects of the pressure-ischemic rabbit eye and found that ischemic damage to the neural retina did not occur in a uniform fashion (Marmot and Dalal, 1993). Instead, they found patchy areas of tissue injury in the neural retina. With regards to the RPE, a transition zone was described between an area of preservation and disruption in an eye exposed to 60 min of ischemia; however, they did not describe the location of this zone. Hughes found an anatomic
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TABLE II
Vertical dimensions of grossly visible retinal lesions produced by 60 rain of retinal ischemia and 58 dabs of reperfusion compared to size of retinal lesions on images acquired after 1 day of reperfusion Superior lesions Eye 1
2 3 mean + S.E.M.
MRI measurement of lesion size* 5-0 4:'3 12"0 7'1 + 2"6
Inferior lesions
Gross measurement of lesion size 4"0 5"3 7"5 5"6 + 1'0
MRI measurement of lesion size 10"4 9"5 10"8 10"2 + 0"4t
Gross measurement of lesion size 7-2 8'0 8'0
7"7 +_0"31-
* All measurements in millimeters. t P = 0-04, t-test. susceptibility of the RPE to pressure-induced ischemia in the rat eye (Hughes, 1991). In eyes that underwent 2 hr of ischemia and 2 weeks of reperfusion, peripheral areas of RPE remained intact while central areas showed extreme attenuation or absence of the RPE. A transition zone was also identified that showed metaplastic changes in the RPE cells. The results of the present study suggest that a different pattern of ischemic damage occurs in the rabbit RPE. Using leakage as an indicator of RPE damage, the most susceptible areas of ischemia were found in the inferior periphery and immediately superior to the medullary ray. This pattern of damage was confirmed by clinical examination as well as, to some extent, by light microscopy. It is not clear as to why certain areas of the outer BRB appear more susceptible to damage t h a n others. One possibility is that the RPE is less able to withstand a given duration of ischemia in certain areas, perhaps because of lower stores of energy substrates or greater accumulation of toxic metabolic by-products. Another explanation concerns the reperfusion period. In various tissues, ischemia-reperfusion results in damage during the reperfusion phase as well as in the ischemic period (McMillen, Huribal and Sumpio, 1993). Reperfusion injury has been attributed to a no-reflow phenomenon resulting from microvascular occlusion by leukocytes. Therefore, it is possible that selected areas of the rabbit choroidal vasculature are more susceptible to the no-reflow p h e n o m e n o n than others. Another aspect of BRB damage in this study concerns its possible relationship to loss of neuroretinal function. Although electroretinographic (ERG) measurements were not done in this study, it is possible to compare the extent of BRB damage (size of the lesion) with the loss of ERG wave amplitudes observed by other investigators in the pressureischemic rabbit eye. The work of Ulrich and Reimann (1986) proved useful in this regard. Their study focused on the b-wave amplitude because it is the most sensitive component of the ERG to ischemia (Foulds and Johnson, 1974). The b-wave amplitudes recorded one day after complete ischemia in their study was
100%, 80%, 2 5 % and 0% of normal after ischemic intervals of 30, 40, 60 and 90 min, respectively. After 20 min of ischemia, 95 % sparing was observed, which is somewhat consistent with the 100% recovery of b-wave amplitude after 30 min of ischemia. After 100 min of ischemia, there was 0 % BRB sparing, which compares reasonably well with the 0 % recovery of b-wave amplitude that was observed following 90 min of ischemia. Therefore, general agreement is observed between the size of BRB lesions and ERG bwave amplitudes. However, quantitative differences should be expected between these endpoints because ERG and contrast-enhanced MR/ provide different types of functional information. In this study, it was not possible to measure inner BRB breakdown with certainty, although the superior leakage found in m a n y eyes was strongly suggestive of its occurrence. In eyes exposed to 20 min of ischemia, focal leakage was seen in several eyes that remained isolated to the area overlying the medullary ray. In one eye exposed to 40 min of ischemia, superior leakage was comprised of two juxtaposed areas of leakage: one very focal area over the medullary ray and another larger area just superior to the first (image not shown). Because the leakage from these areas blended together somewhat on all images, it was analyzed as a single lesion. However, on the basis of location and focality of the leakage, we attributed the first of the two areas of leakage to inner BRB breakdown. Lesion size and distribution identified by M R / o n day 1 after retinal ischemia correlated significantly with measurements of pigmentary lesions obtained from gross ocular specimens 2 months after the ischemia (Table n). However, the m e a n lesion size predicted by M R / w a s larger than the final RPE lesion size. There are two possible explanations for this observation: first, leakage measured on the images was, in fact, larger t h a n the actual area of increased permeability. This might occur when, during the elapsed time of image acquisition, some Gd-DTPA diffuses laterally from the edges of the lesion. In order to minimize this error, lesion sizes were measured from the first post-
556
injection image. Second, the increased area of permeability m a y have included a ' w a t e r s h e d ' area of injured RPE that initially leaked, but was able to retain a normal clinical appearance during the 2-month reperfusion period. We suspect that a combination of these possibilities may have accounted for the overestimation of RPE lesion size. Although quantitative information regarding BRB permeability is not available in this model with which to compare these results, morphologic studies do lend support to some of the findings. Minimal leakage was observed in eyes subjected to 20 min of ischemia, but there was no inferior leakage in these eyes to suggest outer BRB breakdown had occurred. This is consistent with the detailed histologic and ultrastructural studies of Johnson and Foulds who investigated the effects of pressure-ischemia on the retina of the Dutch-belted rabbit (Johnson and Foulds, 1978). They found that an ischemic interval of 15 min produced no acute injury to the RPE. However, 30 min of ischemia or longer caused vacuolization of the RPE cell cytoplasm. More severe changes, which included cellular distortion, were observed after 90 and 1 2 0 m in of ischemia. They also observed microscopic edema of the nerve fiber layer in the medullary ray and vacuolization of endothelial cells in the preretinal blood vessels after only 15 min of ischemia. This lends support to the contention that inner BRB breakdown accounted for the mild leakage observed at the location of the medullary ray after only 20 min of ischemia in the present study. The effect of total ischemia on the BRB could be modulated by a variety of factors, including energy substrate availability, local retinal temperature, state of retinal dark adaptation, and choice and dose of anaesthetics [ketamine has been found to exert a protective effect on the neural retina during ischemia, but its effect on the RPE is not k n o w n (Tsukahara et al., 1992)]. In addition, some authorities have suggested the possibility that mechanical factors related to elevated intraocular pressure could account for some of the tissue damage encountered in this model (Blair et al., 1989; Marmor and Dalai, 1993). However, the absence of any direct evidence of mechanical damage has cast doubt on this explanation (Marmor and Dalal, 1993). An effort to thoroughly address the influence of various modulating factors on injury to the BRB in this model is currently underway in our laboratory. In summary, several biologic questions regarding BRB function in the pressure-ischemic rabbit eye were addressed in this study. First, BRB breakdown is not a uniform phenomenon, but rather a ' p a t c h y ' process involving several predisposed areas. This is in agreement with previous morphologic evidence (Marmor and Dalal, 1993). It becomes more uniform at ischemia durations of 80 min or longer. Secondly, the threshold for BRB damage occurs between 20 and 40 min duration of ischemia. Thereafter, increasing
C.A. W l L S O N E T A L .
the duration of ischemia produces a graded increase in BRB permeability through l O O m i n of ischemia. Finally, BRB breakdown decreases with time after ischemia, but repair is a long process and complete restoration of function m a y not always occur. The relevance of these findings to h u m a n retinal disease remains to be determined. Interruption of blood flow to both the retina and choroid could occur in some instances such as acute glaucoma or ophthalmic artery occlusion, but it is not typical of most ischemic retinopathies in humans. In addition, there are differences in the enzymology of rabbit and h u m a n retinas, particularly with regards to energy snbstrate metabolism (Lowry et al., 1961; Lowry, Roberts and Lewis, 1956; Ames and Li, 1992). Such differences must be considered before extrapolating these results to other species. However, investigations of variables that might influence the susceptibility of the BRB to ischemic injury in the rabbit eye could lead to new treatment strategies, which then could be examined in other animal models.
Acknowledgements Supported by a grant from the Juvenile Diabetes Foundation, Int., New York (Dr Wilson) and an unrestricted grant from Research to Prevent Blindness (RPB), New York. Additional support provided by the National Center for Research Resources and the Biomedical Research Technology Program (BRTP). Dr Berkowitz is the recipient of a RPB career development award. The authors thank ]ackye Lambert for her expert technical assistance. Statistical consultation and analyses were provided by Dr Tom Carmody.
References Ames, A. and Li, Y.Y. (1992). Energy requirements of glutamatergic pathways in rabbit retina. J. Neurosci. 12, 4234-42. Berkowitz, B. A., Sato, Y., Wilson, C. A. and de Juan, E., Jr (1991). Blood-retinal barrier breakdown investigated by real-time magnetic resonance imaging after gadolinium-diethylenetriaminepentaacetic acid injection. Invest. Ophthalmol. Vis. Sci. 32, 2854-2860. Berkowitz, B. A., Torts, P. S., Sen, H. A., Ando, N. and de Juan, E., Jr (1992). Accurate and precise measurement of blood-retinal barrier breakdown using dynamic GdDTPA MRI. Invest. Ophthalmol. Vis. Sci. 33, 3500-6. Blair, N.P., Baker, D.S., Rhode,J. P. and Solomon, M. (1989). Vitreoperfusion: a new approach to ocular ischemia. Arch Ophthalmol 107, 417-23. Cunha-Vaz, J. G. (1979). The blood-ocular barriers. Surv. Ophthalmol. 23, 279-96. Ferris, F.L. and Patz, A. (1984). Macular edema. A complication of diabetic retinopathy. Surv. Ophthalmol. 28, 452-61. Foulds, W.S. and Johnson, N.F. (1974). Rabbit electroretinogram during recovery from induced ischemia. Trans. Ophthalmol. Soc. UK 94, 383-93. Hayreh, S.S., Kolder, H.E. and Weingeist, T.A. (1980). Central retinal artery occlusion and retinal tolerance time. Ophthalmology 87, 75-8. Heike, M. and Marmor, M.F. (1991). Recovery of retinal pigment epithelial function after ischemia in the rabbit. Invest. Ophthalmol. Vis. Sci. 32, 73-7.
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Hughes, W. F. (1991). Quantitation of ischemic damage in the rat retina. Exp. Eye Res. 53, 573-82. Johnson, N. F. and Foulds, W. 8. (1978). The effects of total acute ischemia on the structure of the rabbit retina. Exp. Eye Res. 27, 45-59. Kaskel, D., Valenzuela, H., Hockwin, 0., Muntau, M. and Schedtler, C.-M. (1976). Enzymologic and histologic investigations in normal and pressure-ischemic retina of rabbits. Graefe's Arch. Clin. Exp. Ophthalmol. 200, 71-8. Lowry, O.H., Roberts, N.R. and Lewis, C. (1956). The quantitative histochemistry of the retina. ]. Biol. Chem. 220, 879-92. Lowry, O. H., Roberts, N. R., Schulz, D. W., Clow, J. E. and Clark, J.R. (1961). Quantitative histochemistry of retina. II. Enzymes of glucose metabolism. ]. Biol. Chem. 236, 2 8 t 3 - 2 0 . Marmor, M. F. and Dalai, R. (1993). Irregular retinal and RPE damage after pressure-induced ischemia in the rabbit. Invest. Ophthalmol. Vis. Sci. 34, 2570-5.
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McKechnie, N. M., Johnson, N. F. and Foulds, W. S. (1982). The combined effects of light and acute ischemia on the structure of the rabbit retina: a light and electron microscopic study. Invest. Ophthalmol. Vis. Sci. 22, 449-59. McMillen, M.A., Huribal, M. and Sumpio, B. (1993). Common pathway of endothelial-leukocyte interaction in shock, ischemia, and reperfusion. Am. ]. Surg. 166, 557-562. Tofts, P. S. and Berkowitz, B. A. (1993). Rapid measurement of capillary permeability using the early part of the dynamic Gd-DTPA MRI enhancement curve. ]. Magn. Res. 102, 129-36. Tsukahara, Y., Blair, N.P., Spellman Eappen, D. C., Moy, J.J., Takahashi, A., Shah, G.K. and Viana, M. A. G. (1992). Ketamine suppresses ischemic injury in the rabbit retina. Invest. Ophthalmol. Vis. Sci. 33, 1822-5. Ulrich, W.-D. and Reimann, J. (1986). Survival and revival times of the retina: ERG studies at total ocular ischemia in rabbits and cats. Doc. Ophthalmol. 63, 91-9.