April 2012
Volume 53, Issue 4
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Retina  |   April 2012
Electrophysiological Findings in a Porcine Model of Selective Retinal Capillary Closure
Author Affiliations & Notes
  • Chi D. Luu
    From the 1Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Victoria, Australia; the Singapore Eye Research Institute, Singapore; and the National University of Singapore, Singapore.
  • Wallace S. Foulds
    From the 1Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Victoria, Australia; the Singapore Eye Research Institute, Singapore; and the National University of Singapore, Singapore.
  • Charanjit Kaur
    From the 1Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Victoria, Australia; the Singapore Eye Research Institute, Singapore; and the National University of Singapore, Singapore.
  • Corresponding author: Chi D. Luu, Macular Research Unit, Centre for Eye Research Australia, Level 1, 32 Gisborne Street, East Melbourne, VIC 3002, Australia; cluu@unimelb.edu.au
Investigative Ophthalmology & Visual Science April 2012, Vol.53, 2218-2225. doi:10.1167/iovs.11-8490
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      Chi D. Luu, Wallace S. Foulds, Charanjit Kaur; Electrophysiological Findings in a Porcine Model of Selective Retinal Capillary Closure. Invest. Ophthalmol. Vis. Sci. 2012;53(4):2218-2225. doi: 10.1167/iovs.11-8490.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To determine the effects on the electroretinogram (ERG) of retinal capillary closure induced in the pig by embolization with microspheres.

Methods: Fourteen Yorkshine Landrace pigs of 25- to 45-kg body weight were used. With a customized cannula introduced into the external carotid artery, 10-μm diameter microspheres were delivered to the origin of the vessel that supplies blood to the eye in the pig. Fundus fluorescein angiography and electroretinography were performed between days 7 and 28 post injection. The ERG responses of embolized eyes were compared with those of the contralateral nonembolized eyes.

Results: The amplitudes of the scotopic b-wave (P = 0.002), the maximal b-wave (P < 0.010), the photopic a-wave (P < 0.001) and b-wave (P < 0.001), and the scotopic oscillatory potentials (OPs) (P = 0.025) and photopic OPs (P = 0.036) were significantly reduced in embolized eyes. The reduction of these ERG amplitudes was significantly correlated with the number of microspheres in the retina. There was no significant difference in the combined rod–cone bright flash (maximal) ERG a-wave amplitude between eyes with and without microspheres. Implicit times, however, were similar in embolized and control eyes.

Conclusions: In eyes embolized with microspheres, the amplitudes of most ERG components were significantly reduced without alteration of their implicit times. The magnitude of ERG amplitude reduction correlated with the number of microspheres in the retina.

Introduction
Retinal capillary nonperfusion with resulting retinal hypoxia is a recognized feature of preproliferative diabetic retinopathy, ischemic central retinal vein occlusion (CRVO), and also occurs in the ocular ischemic syndrome (OIS). 
Retinal hypoxia has been identified as a major factor contributing to the development of diabetic retinopathy. 1,2 Retinal hypoxia in the diabetic eye is in part the result of glycation of red cell hemoglobin that increases its oxygen binding capacity with a resulting reduced oxygen supply to affected tissues. 3 Additionally, thickening of capillary basement membrane in the diabetic eyes reduces the caliber of affected capillaries, while hypoxia-induced upregulation of adhesion molecules promotes capillary closure by impacted leukocytes. 4  
Abnormality in components of the electroretinogram (ERG), such as a reduction in the amplitudes of the oscillatory potentials (OPs), has been reported previously as an early sign of retinal dysfunction in diabetes. 510 More recently, a reduction in the amplitudes of the scotopic b-wave, photopic a- and b-wave, and OPs has been reported to occur in diabetic eyes without retinopathy. 11 In eyes with preproliferative diabetic retinopathy, ERG abnormality is likely to result from a combination of a generalized metabolic abnormality in the retina and the effects of retinal capillary closure that is likely to increase the degree of retinal hypoxia known to affect the diabetic eye. 
To assess the effects of retinal capillary closure alone, we investigated the ERG changes that occur as a result of retinal capillary closure induced in the nondiabetic pig eye. They developed a novel model of selective retinal capillary closure induced by embolization of the retina with fluorescent microspheres of 10-μm or 15-μm diameter, 12 each of which exceeds the luminal diameter of retinal capillaries in the pig eye, which have a caliber of 3 to 5 μm. 13  
We have reported preliminary data on the combined rod–cone bright flash ERG in embolized eyes and fellow control nonembolized eyes 12 and have shown a significant reduction in the amplitudes of the ERG b-wave and OPs in embolized eyes. In this article, the authors reported in more detail the effects on the photopic and scotopic ERG of capillary closure induced by retinal embolization with microspheres of 10-μm diameter. 
Methods
All experiments were carried out in the SingHealth Experimental Medicine Centre (SEMC) located on the campus of the Singapore General Hospital. All surgical procedures were performed in a properly equipped surgical operating theater in sterile conditions and were approved by the Institutional Animal Care and Use Committee of the SEMC. The SEMC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Animals
Yorkshine Landrace pigs of 25- to 45-kg body weight were used in this study. Animals were sedated with ketamine intramuscularly (10 mg/kg body weight) and anesthetized with a 5% to 10% halothane/oxygen mixture for induction and a 2% mixture for maintenance, delivered via an endotracheal tube. Heart rate and blood pressure were monitored and oxygenation assessed by ear oximetry. Pupils were dilated with 3 drops of tropicamide 1% (Alcon Laboratories, Inc., Fort Worth, TX) instilled into each eye at 5-minute intervals. 
Injection of Microspheres
The method of delivering microspheres into the retina has been described in detail previously. 12 In brief, one carotid artery was exposed in the neck and the dissection carried upward to expose the external carotid artery. A customized cannula with a closed end and a side port 10 mm from the closed tip was inserted into the external carotid artery via a small opening in the arterial wall and advanced until the closed tip engaged in the narrowing lumen of the inferior orbital artery, thus preventing onward dissemination into more distal tissues including the fellow nonembolized eyes. At this point, it was estimated that the side port was in the vicinity of the origin of the external ophthalmic artery that, in the pig, supplies the eye and orbit with blood. Three milliliters of a suspension of microspheres of 10-μm diameter, made up to 5 mL with sterile normal saline, was injected via the cannula over a period of 3 to 5 seconds, followed by a washout injection of 10 mL of sterile normal saline. After the injection of the microspheres, the cannula was removed and the opening in the wall of the external carotid artery was closed with 10-0 nylon sutures. The neck wound was then closed in layers and skin closure completed with a continuous subcuticular suture. The ocular fundus of the relevant eye was then examined with a fundus camera fitted with filters for fluorescein angiography, to confirm that the fluorescent microspheres were visible. 
After wound closure, the animals were given a prophylactic intramuscular injection of a combined ampicillin/cloxacillin antibiotic (Ampliclox 100 mg; GlaxoSmithKline, Uxbridge, UK) and allowed to recover under close observation in the SEMC. The animals were allowed to survive for between 7 and 28 days, and electrophysiology and fluorescein angiography were performed in all animals before euthanasia induced in anesthetized animals by an overdose of pentobarbitone administered intramuscularly. Our previous results showed that the microspheres detectable in the retina immediately after delivery were still present in the same locations in the retina at the various survival time intervals before euthanasia up to 28 days after delivery. 
Retinal Imaging
Fundus fluorescein angiograms (FFAs) of 50° centered on the optic disc were captured with a digital fundus camera (TRC-50EX; Topcon, Tokyo, Japan), after an injection of 0.5 mL sodium fluorescein solution (15%) was administered via an ear vein. The FFA images were used for estimating the number of microspheres in the retina. 
Electrophysiology
Full-field ERGs were recorded with a corneal bipolar Burian-Allen electrode (Hansen Ophthalmic Development Labs, Coralville, IA). A custom-made platinum wire electrode was inserted through one ear and served as the ground electrode. Stimuli were brief white flashes (4 ms) delivered through a mini-Ganzfeld sphere (ColorBust; Diagnosis LLC, Lowell, MA). The Espion system (Espion; Diagnosis LLC) was used for stimulus generation and data acquisition. 
The recording protocol used was based on, and extended from, the International Society for Clinical Electrophysiology of Vision standard for human clinical full-field ERG. 14,15 The protocol consisted of 30 minutes of dark-adaptation, followed by recording of a dark-adapted intensity series by using white flash stimuli ranging from −4.0 log cd·s·m−2 to 1.0 log cd·s·m−2. The interval between flashes was initially set at 5 ms and progressively lengthened to 20 ms as the stimulus intensity increased. 
After completion of the dark-adapted intensity series, animals were light adapted for 10 minutes to a steady white background of 30 cd·m−2 with the same Ganzfeld stimulator. Light-adapted intensity series were recorded for intensities between −1.3 log cd·s·m−2 to 1.0 log cd·s·m−2. The interstimulus interval was set at 2 ms for low flash intensities and progressively increased to 10 ms as the stimulus intensity increased. ERGs from only one eye were recorded at a time and there was no particular order in which each of the eyes was tested first. The ERG result at each intensity level was obtained by averaging at least 5 responses. The ERG of each animal was recorded at one time point immediately before euthanasia. 
Analysis
ERG parameters (response amplitudes and implicit times of various components) were obtained in both scotopic and photopic conditions. A Student's t-test was used to determine the level of difference in ERG parameters between eyes with and without microspheres. The maximum response amplitude of the scotopic b-wave (R max) and the retinal sensitivity (k, the flash intensity that produces half R max) were calculated by using a Naka-Rushton function as described previously by others. 1619 A least-squares minimization procedure was used to fit the Naka-Rushton curve to the lower limb of the intensity-response data. Curve fitting was achieved with a computer spreadsheet (Excel; Microsoft, Redmond, WA) and all parameters (R max, k, and the slope of the function “n”) were free to vary. 
An estimate of the number of microspheres in the retina was obtained from FFAs taken before euthanasia. Following embolization, we found on histology 12 that microspheres in the retina were confined to capillaries in the nerve fiber layer of the inner retina. With FFAs focused on the inner retina, images of microspheres in the retina were sharp, while those in the choroid were blurred and of a much lower intensity than microspheres in the retina (Fig. 1A). As previously reported, 12 although a large number of microspheres passed through the choroid to reach the systemic circulation, the presence of microspheres in the fellow eye was an extremely rare event and was not present in any of the fellow eyes used as controls in the present study. As the microspheres were fluorescent, with absorption and emission characteristics similar to those of sodium fluorescein, they were easily detectable on FFA. As it seemed likely that the number of microspheres detectable on FFA would provide a good indication of the extent of the resulting capillary closure, FFA images were used to quantify the number of retinal microspheres within the imaged area of the fundus. To achieve this, we used, first, the threshold function in Adobe Photoshop (Adobe Systems Inc., San Jose, CA) to remove background information including the retinal vasculature, optic nerve head, and out-of-focus microspheres located within the choroid (Fig. 1B). We then applied the particle analysis procedure available from Igor software (WaveMetrics, Portland, OR) to count the number of identified microspheres (Fig. 1C). This procedure sequentially ascribed a number to each bright spot of a predetermined size. This method provided an effective and reliable assessment of the number of microspheres within retinal capillaries in the imaged area and thus, an estimate of the extent of retinal capillary embolization. To determine if the number of microspheres in the retina correlated with histologic findings, the relationship between retinal thickness and number of microspheres was examined. Measurements of retinal thickness were made from histologic sections as previously described. 12 To account for the variation in retinal thickness among animals of different ages and weights, the number of microspheres were compared with the retinal thickness ratio between embolized and nonembolized fellow control eye. The relationship between the differences in the amplitudes of the various recorded ERG parameters in control eyes without microspheres and in eyes with microspheres and the number of microspheres in embolized eyes was analyzed by using Pearson's correlation. 
Figure 1.
 
(A) Fundus fluorescein angiogram (50° view) showing microspheres and retinal vasculature. Note that microspheres located in the retinal capillaries appear sharp and much brighter than microspheres located in the choroid. (B) Only retinal microspheres are visible after the threshold function was applied. (C) Particle analysis procedure was used for estimating the number of microspheres by sequentially ascribing a number to each bright spot.
Figure 1.
 
(A) Fundus fluorescein angiogram (50° view) showing microspheres and retinal vasculature. Note that microspheres located in the retinal capillaries appear sharp and much brighter than microspheres located in the choroid. (B) Only retinal microspheres are visible after the threshold function was applied. (C) Particle analysis procedure was used for estimating the number of microspheres by sequentially ascribing a number to each bright spot.
Results
ERG data from 14 animals with successfully delivered microspheres to one eye were presented in this article. The typical dark-adapted full-field ERG responses to various stimulus intensities in a nonembolized eye, and the group average of the intensity-response (IR) function for a- and b-wave in embolized and nonembolized eyes are shown in Figure 2. The IR function of the scotopic a-wave was similar in eyes with and without microspheres. The IR function of the scotopic b-wave was significantly reduced in eyes with microspheres, compared to that of control eyes, particularly at higher flash intensities. 
Figure 2.
 
Left: Representative response wave forms of the dark-adapted (scotopic) ERG with various flash intensities in a nonembolized eye. Right: The intensity-response (IR) function for the scotopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). The difference in IR function between eyes with and without microspheres was significant for the scotopic b-wave (especially at high-intensity levels) but not for the a-wave. Error bars of IR function represent 95% confidence intervals.
Figure 2.
 
Left: Representative response wave forms of the dark-adapted (scotopic) ERG with various flash intensities in a nonembolized eye. Right: The intensity-response (IR) function for the scotopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). The difference in IR function between eyes with and without microspheres was significant for the scotopic b-wave (especially at high-intensity levels) but not for the a-wave. Error bars of IR function represent 95% confidence intervals.
A typical fitting of the Naka-Rushton curve to the IR data is shown in Figure 3. The maximum b-wave amplitude (R max) of embolized eyes (493.9 ± 117.9 μV) was significantly reduced as compared to that of the nonembolized eyes (604.2 ± 56.2 μV, P < 0.001). There was no significant difference in log (k) between embolized eyes (−2.335 ± 0.167) and nonembolized eyes (−2.247 ± 0.082, P = 0.112). 
Figure 3.
 
A typical fitting of the Naka-Rushton curve (black line) to the intensity-response data (grey dots) of a control eye.
Figure 3.
 
A typical fitting of the Naka-Rushton curve (black line) to the intensity-response data (grey dots) of a control eye.
Typical light-adapted full-field ERG responses to various stimulus intensities and the group average of the IR functions for photopic a- and b-wave are shown in Figure 4. Unlike the IR function of the scotopic ERG, the IR functions of both photopic a-wave and b-wave were significantly reduced in eyes with microspheres, compared with those of the contralateral nonembolized control eyes, especially at higher flash intensity levels. 
Figure 4.
 
Left: Representative response wave forms of the light-adapted (photopic) ERG in nonembolized eyes at various flash intensities. Right: The IR functions for the photopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). On average, the IR function of both photopic a-wave and b-wave of eyes with microspheres was significantly reduced as compared to that of eyes without microspheres. Error bars of the IR function represent 95% confidence intervals.
Figure 4.
 
Left: Representative response wave forms of the light-adapted (photopic) ERG in nonembolized eyes at various flash intensities. Right: The IR functions for the photopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). On average, the IR function of both photopic a-wave and b-wave of eyes with microspheres was significantly reduced as compared to that of eyes without microspheres. Error bars of the IR function represent 95% confidence intervals.
The mean response amplitudes and implicit times of the scotopic a- and b-wave at flash intensities of 0.01 and 10 cd·s·m−2 for eyes with and without microspheres are presented in Table 1. There were no significant differences in implicit times between eyes with and those without microspheres. The mean response amplitudes and implicit times of the photopic a- and b-wave at a flash intensity of 3 cd·s·m−2 for eyes with and without spheres are also presented in Table 1. Similarly to the implicit times of the scotopic ERG, none of the implicit times of the photopic ERG was significantly different between eyes with and those without microspheres. 
Table 1.
 
ERG Parameters of Eyes with and without Microspheres
Table 1.
 
ERG Parameters of Eyes with and without Microspheres
Parameters Without Spheres (Mean ± SD) With Spheres (Mean ± SD) P Value
Response amplitude (μV)
 Scotopic b-wave (0.01 cd·s·m−2) 396.4 ± 43.7 326.6 ± 78.3 0.002
 Maximal a-wave (10 cd·s·m−2) 329.3 ± 30.9 304.4 ± 63.4 0.128
 Maximal b-wave (cd·s·m−2) 796.0 ± 77.4 681.9 ± 164.8 0.010
 Scotopic OP (10 cd·s·m−2) 466.6 ± 136.3 352.7 ± 58.2 0.025
 Photopic a-wave (3 cd·s·m−2) 51.4 ± 5.7 38.5 ± 8.9 <0.001
 Photopic b-wave (3 cd·s·m−2) 519.7 ± 36.9 436.9 ± 81.3 <0.001
 Photopic OP (3 cd·s·m−2) 249.8 ± 42.8 204.7 ± 45.0 0.036
Implicit time (ms)
 Scotopic b-wave (0.01 cd·s·m−2) 83.3 ± 4.3 82.4 ± 5.9 0.442
 Maximal a-wave (10 cd·s·m−2) 12.7 ± 0.5 12.6 ± 0.5 0.750
 Maximal b-wave (10 cd·s·m−2) 45.0 ± 5.0 44.6 ± 4.0 0.884
 Scotopic OP (10 cd·s·m−2) 174.1 ± 8.6 178.9 ± 8.0 0.215
 Photopic a-wave (3 cd·s·m−2) 13.4 ± 0.9 13.5 ± 0.8 0.631
 Photopic b-wave (3 cd·s·m−2) 29.6 ± 1.4 30.0 ± 1.5 0.556
 Photopic OP (3 cd·s·m−2) 205.4 ± 19.3 209.3 ± 19.9 0.657
It has previously been shown that an increase in retinal thickness occurs in embolized eyes as compared with nonembolized fellow eyes, and retinal thickness measurements on histologic sections appear to be good indicators of the degree of retinal hypoxia induced by embolization. 12 There was a significant correlation between the number of microspheres and the retinal thickness ratio measured on histologic sections (r = 0.731, P = 0.025, Fig. 5). 
Figure 5.
 
Relationship between retinal thickness ratio (ratio of thickness in embolized eyes and in nonembolized fellow eyes) and number of microspheres. Lines represent the mean and 95% confidence intervals of the slope.
Figure 5.
 
Relationship between retinal thickness ratio (ratio of thickness in embolized eyes and in nonembolized fellow eyes) and number of microspheres. Lines represent the mean and 95% confidence intervals of the slope.
The relationships between the number of identified microspheres and the differences in the amplitudes of various ERG components between control and embolized eyes are shown in Figure 6. There was a significant correlation between the number of microspheres and the differential amplitudes of the scotoptic b-wave, maximal b-wave, photopic a- and b-wave, and scotopic and photopic OPs. The correlation between the number of microspheres and the differences in the amplitudes of the maximal a-wave between the two groups of eyes was not significant (r = 0.022, P = 0.944). 
Figure 6.
 
Correlation between the differential ERG amplitudes (difference between control and embolized eyes) and number of microspheres in the retina. All ERG components except the maximal a-wave amplitude were significantly correlated with the number of microspheres. Lines of each graph represent the mean and 95% confidence intervals of the slope.
Figure 6.
 
Correlation between the differential ERG amplitudes (difference between control and embolized eyes) and number of microspheres in the retina. All ERG components except the maximal a-wave amplitude were significantly correlated with the number of microspheres. Lines of each graph represent the mean and 95% confidence intervals of the slope.
Discussion
This article reported that in the nondiabetic pig eye, retinal embolization with microspheres of a size capable of occluding retinal capillaries (and some precapillary arterioles), but too small to occlude larger retinal arterioles or choroidal capillaries, caused a significant reduction in the amplitudes of almost all ERG responses. The amplitudes of the scotopic b-wave, the maximal b-wave, the photopic a- and b-wave, and the scotopic and photopic OPs were all significantly reduced in eyes embolized with microspheres, compared to fellow eyes without microspheres. Interestingly, similar ERG changes have recently been reported in diabetic patients without retinopathy. 11  
Pig and human eyes share many anatomic similarities that make the former useful in ophthalmic research. 20,21 Pig eyes have been used as models for the study of the pathogenesis of retinal diseases, the testing of ophthalmic treatments, and surgical procedures. 2228 An advantage of this novel pig model of retinal capillary closure is its unilaterality, for the closed end of the delivery catheter blocks the onward transmission of microspheres and prevents their systemic dissemination. Microspheres were absent from all the fellow control eyes reported here, thus enabling comparisons to be made between embolized and nonembolized eyes in the same animal. The capillary closure induced by retinal embolization with microspheres, as previously reported, lasts for at least as long as 28 days. 12 The authors have also shown that the increase in retinal thickness that develops in embolized eyes persists unchanged during the first 28 days after injection of microspheres. 12  
There is little doubt that occlusion of retinal capillaries is likely to result in reduced oxygen supply to those layers of the retina that receive their oxygenation from arterial blood-carrying capillaries in the inner retina that, in the pig, lie in the nerve fiber layer of the retina. The deeper capillary nets in this species carry venous blood. There was no detectable sign of retinal ischemia on ophthalmoscopy of embolized eyes or on retinal photography. In particular, none of the embolized eyes showed the presence of cotton-wool spots that might have been expected if embolization had occluded a significant number of precapillary arterioles. In spite of this, there were significant morphologic abnormalities in the retinas of embolized eyes on light and electron microscopy. 12  
These morphologic changes included extracellular and intracellular edema with vacuolation and mitochondrial disruption in ganglion cells and Müller cells and abnormalities in the inner nuclear and inner plexiform layers of the retina. 12 The morphology of the outer plexiform layer and outer nuclear layer, together with the photoreceptors and retinal pigment epithelium (RPE), all of which receive their oxygen supply from the choroid, remained normal. 12 The morphologic alterations seen in ganglion cells and Müller cells in embolized eyes were similar to previously reported appearances in hypoxic/ischemic retina. 29  
It is well known that the choroid supplies oxygen to the outer retina, most of which is used by the highly metabolic photoreceptors, while capillaries in the inner retina supply oxygen to the inner and mid-retina. The literature on the distribution of oxygen across the retina in various species has been extensively reviewed by Wangsa-Wirawan and Linsenmeier. 30 In holangiotic retina, such as the pig retina, the inner retina as far as the inner nuclear layer is dependant for its oxygen supply on the capillary circulation in the nerve fiber layer of the retina, while the choroid provides the oxygen supply to the outer retina, especially the highly metabolizing photoreceptors. Depending on light or dark-adapted conditions, there is a variable contribution to the oxygenation of the mid-retina from the overlapping contributions of the inner retinal and choroidal circulations. It is no surprise, therefore, that occlusion of a proportion of inner retinal capillaries resulted in mitochondrial and cytoplasmic damage to ganglion cells and Müller cells, the abnormalities in the latter cells providing an explanation for the reduction in the amplitudes of the scotopic and photopic b-waves and the scotopic and photopic OPs in embolized eyes. 
The findings of the scotopic and photopic a-wave amplitudes are intriguing. A possible explanation for the amplitude reduction observed in the photopic a-wave but not in the scotopic (maximal) a-wave in embolized eyes is that there is a separate pathway for rod and cone visual pigment regeneration. The RPE plays a critical role in visual pigment regeneration for the rods but not the cones. 31 Thus, the scotopic a-wave amplitude, largely derived from rod photoreceptors, would be expected to be normal, as was found in embolized eyes because the choroidal circulation was unaffected by embolization, the injected microspheres being too small to occlude the wider bore capillaries of the choroid. As a result, there was an absence of overt morphologic damage to the RPE following retinal capillary embolization. The selective reduction of the photopic a-wave amplitude, predominantly derived from cone photoreceptors, may be explained by the morphologic and functional alterations of the Müller cells in embolized eyes, for Müller cells have been shown to play a role in cone visual pigment regeneration. 3235 Further studies are required to confirm this observation. A small reduction in the scotopic a-wave amplitude in the embolized eyes observed at a flash intensity of 1.0 log cd·s·m−2 is believed to be associated with a reduction in cone photoreceptor function. 
We and others have shown that the ERG implicit times, particularly the OPs implicit time, is very sensitive to changes in retinal circulation.9,10,36 For example, implicit time delays have been reported to occur early in the diabetic eye before retinopathy is detected clinically, 5,8,37 and to become more abnormal as retinopathy progresses. 38 Implicit times have also been regarded as predictors of disease progression in CRVO 3941 and OIS 42 and to correlate with aqueous levels of vascular endothelial growth factor in CRVO.44 In embolized eyes, however, mean implicit times were not increased as compared with control eyes. In a subgroup of embolized eyes with a large number of microspheres, however, there were increases in implicit times of up to 4 ms. These findings suggest that certain levels of capillary closure by the microspheres need to be achieved to have an effect on the implicit times in this model. 
It is unlikely that the changes in the ERGs observed in this study were due to temporary closure of the carotid artery during injection of microspheres. We have previously shown that in pigs in which a temporary closure of the carotid artery occurred without successful delivery of microspheres to the eye, the ERG responses of eyes in which embolization failed were similar to those of contralateral control eyes one week after attempted embolization. 12  
It is possible that although there was a correlation between the number of microspheres identified in the retina of embolized eyes and most ERG parameters, the degree of ischemia induced in the retina may have been underestimated in counts of retinal microspheres. Firstly, as only FFAs centered on the optic disc and limited to a 50° field of view were used for estimating the number of microspheres in the retina, the number of retinal microspheres reported in this article would be less than the total number of microspheres present in the whole retina. Secondly, we have previously shown in embolized eyes that some microspheres occluded precapillary arterioles with a greater degree of downstream capillary closure than occasioned by microspheres occluding only individual capillaries. As the method used to quantify the number of microspheres detectable in the retina did not differentiate between capillary and precapillary arteriolar occlusion, this additionally might have resulted in an underestimation of the retinal ischemia resulting from embolization with microspheres. Certainly the effects of embolization on retinal morphology and on the ERGs were greater than might have been expected from the number of microspheres detectable in the retina. Direct measurement of retinal oxygen levels between embolized and nonembolized eyes would be necessary to determine if microsphere counts underestimate the degree of ischemia induced by capillary embolization. 
In relation to whether the embolized pig model of retinal capillary closure is also a useful model of capillary closure in the diabetic eye, an obvious difference between these two situations is that in the diabetic eye there are changes in the metabolism of retinal neural and glial cells as a result of the underlying diabetes that do not accompany the capillary closure induced in the nondiabetic pig eye. The model is therefore a model of the capillary closure that can occur in the diabetic eye and other vascular occlusive conditions but not a model of diabetic retinopathy. 
In terms of the actual capillary closure, although a very large number of microspheres were injected, the proportion of injected microspheres reaching the retina was relatively small, some being distributed throughout other tissues in the orbit and a large number passing through the choroid into the venous circulation. None of these microspheres in the venous circulation resulted in any detectable adverse effect and in the present study, as already indicated, no microspheres were detectable in fellow eyes. In flat retinal preparations and on FFA, microshpheres in the retina were randomly and widely distributed throughout the capillaries of the inner retina and only rarely occluded neighboring capillaries (data not shown). As a result, affected areas of retina continued to be supplied with blood from neighboring patent capillaries and no evidence of anoxic cell death was present on electron microscopy sections of embolized retinas. Some reduction in oxygen availability undoubtedly occurred and would explain the morphologic changes short of cell death affecting Müller and ganglion cells, which were previously reported. 12 In this respect, the effects of induced capillary closure resemble those in diabetes where oxygen diffusion from neighboring nonoccluded capillaries prevents anoxic cell death and allows cells in nonperfused areas of retina to survive, although probably functioning subnormally. 
In conclusion, the authors reported a reduction in the amplitudes of the scotopic b-wave, photopic a- and b-wave, and scotopic and photopic OPs of the full-field ERG in an animal model of retinal capillary closure. The extent of capillary closure in embolized eyes was sufficient to cause hypoxic damage to ganglion cells and Müller cells, abnormality in the latter explaining the ERG findings. Among possible explanations is the adverse effect of hypoxia-induced alterations in the cytoplasmic morphology of Müller cells. 
Acknowledgments
The authors thank In Chin Song for assistance with the surgery and other staff of the SingHealth Experimental Medicine Centre for care and monitoring of animals. The authors also thank Wee Kuan Kek for his assistance with the experiment. 
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Footnotes
 Supported by Grant BMRC:05/1/35/19/422 from the Singapore Biomedical Research Council. The Centre for Eye Research Australia receives operational infrastructure support from the Victorian Government.
Footnotes
 Disclosure: C.D. Luu, None; W.S. Foulds, None; C. Kaur, None
Figure 1.
 
(A) Fundus fluorescein angiogram (50° view) showing microspheres and retinal vasculature. Note that microspheres located in the retinal capillaries appear sharp and much brighter than microspheres located in the choroid. (B) Only retinal microspheres are visible after the threshold function was applied. (C) Particle analysis procedure was used for estimating the number of microspheres by sequentially ascribing a number to each bright spot.
Figure 1.
 
(A) Fundus fluorescein angiogram (50° view) showing microspheres and retinal vasculature. Note that microspheres located in the retinal capillaries appear sharp and much brighter than microspheres located in the choroid. (B) Only retinal microspheres are visible after the threshold function was applied. (C) Particle analysis procedure was used for estimating the number of microspheres by sequentially ascribing a number to each bright spot.
Figure 2.
 
Left: Representative response wave forms of the dark-adapted (scotopic) ERG with various flash intensities in a nonembolized eye. Right: The intensity-response (IR) function for the scotopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). The difference in IR function between eyes with and without microspheres was significant for the scotopic b-wave (especially at high-intensity levels) but not for the a-wave. Error bars of IR function represent 95% confidence intervals.
Figure 2.
 
Left: Representative response wave forms of the dark-adapted (scotopic) ERG with various flash intensities in a nonembolized eye. Right: The intensity-response (IR) function for the scotopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). The difference in IR function between eyes with and without microspheres was significant for the scotopic b-wave (especially at high-intensity levels) but not for the a-wave. Error bars of IR function represent 95% confidence intervals.
Figure 3.
 
A typical fitting of the Naka-Rushton curve (black line) to the intensity-response data (grey dots) of a control eye.
Figure 3.
 
A typical fitting of the Naka-Rushton curve (black line) to the intensity-response data (grey dots) of a control eye.
Figure 4.
 
Left: Representative response wave forms of the light-adapted (photopic) ERG in nonembolized eyes at various flash intensities. Right: The IR functions for the photopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). On average, the IR function of both photopic a-wave and b-wave of eyes with microspheres was significantly reduced as compared to that of eyes without microspheres. Error bars of the IR function represent 95% confidence intervals.
Figure 4.
 
Left: Representative response wave forms of the light-adapted (photopic) ERG in nonembolized eyes at various flash intensities. Right: The IR functions for the photopic a- and b-wave in embolized eyes (black) and nonembolized eyes (grey). On average, the IR function of both photopic a-wave and b-wave of eyes with microspheres was significantly reduced as compared to that of eyes without microspheres. Error bars of the IR function represent 95% confidence intervals.
Figure 5.
 
Relationship between retinal thickness ratio (ratio of thickness in embolized eyes and in nonembolized fellow eyes) and number of microspheres. Lines represent the mean and 95% confidence intervals of the slope.
Figure 5.
 
Relationship between retinal thickness ratio (ratio of thickness in embolized eyes and in nonembolized fellow eyes) and number of microspheres. Lines represent the mean and 95% confidence intervals of the slope.
Figure 6.
 
Correlation between the differential ERG amplitudes (difference between control and embolized eyes) and number of microspheres in the retina. All ERG components except the maximal a-wave amplitude were significantly correlated with the number of microspheres. Lines of each graph represent the mean and 95% confidence intervals of the slope.
Figure 6.
 
Correlation between the differential ERG amplitudes (difference between control and embolized eyes) and number of microspheres in the retina. All ERG components except the maximal a-wave amplitude were significantly correlated with the number of microspheres. Lines of each graph represent the mean and 95% confidence intervals of the slope.
Table 1.
 
ERG Parameters of Eyes with and without Microspheres
Table 1.
 
ERG Parameters of Eyes with and without Microspheres
Parameters Without Spheres (Mean ± SD) With Spheres (Mean ± SD) P Value
Response amplitude (μV)
 Scotopic b-wave (0.01 cd·s·m−2) 396.4 ± 43.7 326.6 ± 78.3 0.002
 Maximal a-wave (10 cd·s·m−2) 329.3 ± 30.9 304.4 ± 63.4 0.128
 Maximal b-wave (cd·s·m−2) 796.0 ± 77.4 681.9 ± 164.8 0.010
 Scotopic OP (10 cd·s·m−2) 466.6 ± 136.3 352.7 ± 58.2 0.025
 Photopic a-wave (3 cd·s·m−2) 51.4 ± 5.7 38.5 ± 8.9 <0.001
 Photopic b-wave (3 cd·s·m−2) 519.7 ± 36.9 436.9 ± 81.3 <0.001
 Photopic OP (3 cd·s·m−2) 249.8 ± 42.8 204.7 ± 45.0 0.036
Implicit time (ms)
 Scotopic b-wave (0.01 cd·s·m−2) 83.3 ± 4.3 82.4 ± 5.9 0.442
 Maximal a-wave (10 cd·s·m−2) 12.7 ± 0.5 12.6 ± 0.5 0.750
 Maximal b-wave (10 cd·s·m−2) 45.0 ± 5.0 44.6 ± 4.0 0.884
 Scotopic OP (10 cd·s·m−2) 174.1 ± 8.6 178.9 ± 8.0 0.215
 Photopic a-wave (3 cd·s·m−2) 13.4 ± 0.9 13.5 ± 0.8 0.631
 Photopic b-wave (3 cd·s·m−2) 29.6 ± 1.4 30.0 ± 1.5 0.556
 Photopic OP (3 cd·s·m−2) 205.4 ± 19.3 209.3 ± 19.9 0.657
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