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Retinal Vessel Changes in Galactose-Fed Dogs
Tatsuji Kobayashi, MD;
Eri Kubo, MD;
Yukio Takahashi, MD, PhD;
Toshiharu Kasahara, PhD;
Hidetoshi Yonezawa, PhD;
Yoshio Akagi, MD, PhD
Arch Ophthalmol. 1998;116:785-789.
ABSTRACT
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Background Retinal lesions similar to those in human early-stage diabetic retinopathy have been reported to occur in dogs fed galactose for long periods. Investigations of retinal changes, however, have been limited to studies of the intact retinal vasculature isolated by trypsin digestion.
Objective To document the onset and progression of retinal lesions in galactose-fed dogs by the common clinical techniques of fundus color photography and fluorescein angiography.
Methods Fourteen 6-month-old male beagles made aphakic in 1 eye were divided into a control group (4 dogs), receiving a diet containing 30% cellulose, and a galactosemic group (10 dogs), receiving a diet containing 30% galactose. The progression of retinal changes in these dogs was periodically monitored by color fundus photography and fluorescein angiography.
Results Dogs fed a 30% galactose diet for 28 to 41 months were observed by fluorescein angiography and color fundus photography to develop, in order of frequency, microaneurysms, retinal hemorrhages, intraretinal microvascular abnormalities, retinal nonperfused areas, and varicose and serpiginous veins. These findings are similar to the early clinical retinal changes observed in humans with diabetes.
Conclusion These results confirm that galactosemic dogs are an appropriate and suitable animal model for investigating human diabetic retinopathy.
INTRODUCTION
THE OBSERVATION that dogs fed galactose for long periods develop retinal lesions that are similar to those observed in humans with diabetes has attracted widespread attention to this animal model of human diabetic retinopathy.1-3 This model develops all stages of retinal lesions, ranging from initial background retinopathy to the proliferative stage. Histological studies of intact retinal vessels isolated by trypsin digestion indicate that the lesion that initiates these retinal changes is the selective degeneration of retinal capillary pericytes.4-6 The progression observed in these histological studies has now been confirmed using the common clinical technique of fluorescein angiography in galactose-fed dogs. Because galactose feeding results in sugar cataract formation, dogs in the present study were made aphakic in 1 eye prior to the administration of the galactose chow so that the inevitable formation of sugar cataracts would not interfere in periodic retinal examinations and fluorescein angiography.7
MATERIALS AND METHODS
DOGS
All animal studies conformed to the Association for Research in Vision and Ophthalmology resolution on the treatment of animals. Fourteen 6-month-old male beagles were obtained (CSK Research Park Inc, Nagano, Japan). Prior to the onset of the study, all dogs were made aphakic in 1 randomly chosen eye by phacoemulsification. Two days prior to the lensectomy, antibiotics (0.3% micronomicin sulfate solution) were topically applied twice daily to both eyes. Dogs were anesthetized with an intravenous injection of 25 mg/kg of sodium pentobarbital. The clear lens was asceptically removed by phacoemulsification and aspiration. This was preceded by mydriais with topical administration of a mixture of 0.5% tropicamide hydrochloride, 0.5% phenylephrine hydrochloride, and 0.1% epinephrine. After the operation, 0.3% micronomicin and 0.1% betamethasone sodium phosphate were topically applied 3 times daily to the operated eye for 3 weeks.
After the lens extraction, all dogs were divided into 2 groups. The control group (4 dogs) was fed a control diet containing 30% cellulose and the galactose-fed group (10 dogs) was fed a diet containing 30% galactose. Serum glucose and galactose concentrations and erythrocyte galactitol levels in these dogs were measured at 6-month intervals. Blood samples were obtained in the early morning before feeding.
OPHTHALMIC EXAMINATION OF RETINAL LESIONS
Fluorescein angiography was conducted at 4-week intervals beginning at 26 months after the initiation of experimental diets. Dogs were administered 2 mL of 10% fluorescein sodium via the foreleg vein. Fluorescein angiograms and fundus color photographs were obtained in unanesthetized dogs using a handheld direct-image retinal camera (Genesis, Kowa Co Ltd, Tokyo, Japan). Two types of filters, an excitation filter (460-510 nm) and a barrier filter (530-650 nm), were used in the fluorescein angiograms.
RESULTS
Dogs were fed the appropriate diet for 41 months and the retinas in all aphakic eyes were examined at 4-week intervals, beginning at 26 months of galactose feeding. One dog in the galactose-fed group died as a result of the excessive administration of intravenous anesthetics; thus, fundus examinations were limited to 4 control dogs and 9 galactose-fed dogs. No significant differences between the control or galactose-fed dogs were observed in either blood glucose levels (Table 1) or other hematological test results, serum electrolyte levels, urinalysis results on fresh urine, or urinalysis results on cumulative urine. Both plasma galactose and galactitol concentrations were elevated in the galactose-fed group but not in the control group (Table 1). Fluorescein angiography was conducted at 4-week intervals beginning at 26 months after the initiation of experimental diets. Unlike the X pattern seen in humans, the main retinal vessels in the normal control dog are elongated in a reverse T pattern centering around the optic disc. A triangular white reflecting plate referred to the tapetum is seen in the upper half of the posterior pole of the eyeball. The first retinal lesion detected by fluorescein angiography was the appearance of a small punctate hyperfluorescence accompanied by the faint leakage of fluorescein after 28 months of galactose feeding (Figure 1, A). These corresponded to the appearance of small retinal dot and blot hemorrhages representing leaky microaneurysms in color fundus photographs. The progression of retinal lesions at the same locus was precisely monitored for the next 10 months until the 38th month of galactose feeding. Two months after the appearance of the initial lesion (30 months after the onset of galactose feeding), fluorescein leakage from this area disappeared and perfusion of fluorescein dye progressively decreased, indicating the formation of a small area of nonperfusion (Figure 1, B). After 31 months of galactose feeding, multiple punctate and spotted leakage of fluorescein corresponding to the appearance of dot and blot hemorrhages appeared, and increased permeability of the capillaries was observed at the periphery of the small nonperfused area (Figure 1, C). Two months later (33 months of galactose feeding), the areas previously demonstrating multiple punctate spotted fluorescein leakage and increased capillary permeability had developed into a larger area of nonperfusion (Figure 2, A). After 35 months of galactose feeding, the area of nonperfusion became larger by the confluence of a small, nonperfused area observed at 33 months of feeding (Figure 2, B). Blockages of background fluorescence were observed in some areas as black dots (indicated by the arrows in Figure 1, C), and these retinal changes were confirmed by the appearance of spotted retinal hemorrhages on the fundus color photographs taken during the same period (Figure 3). Finally, by 39 months of galactose feeding, a broad area of nonperfusion had formed by the repetitive confluence of nonperfused areas previously demonstrating fluorescein leakage and hyperpermeability of retinal capillaries (Figure 2, C). Blood supply in most of the large retinal arterioles demonstrating severe capillary closure showed obstructed blood flow (Figure 2, B, arrow) and the appearance of sausagelike dilatation and serpiginous veins (Figure 2, C, arrow).
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Table 1. Glucose and Galactose Levels*
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Figure 1. Progression of retinal lesions in the fluorescein angiogram of a dog retina (dog 208) from the galactose-fed group. A, Spotted hyperfluorescence with late leakage (which should be called putative microaneurysm) first could be detected at 28 months of feeding. B, Fluorescein leakage at this area disappeared (arrows) at 30 months. Perfusion of dye in this area progressively decreased. C, Multiple occurrences of punctate and spotted leakage of fluorescence at the area surrounding the previously developed small nonperfusion area could be seen at 31 months.
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Figure 2. A, The areas revealing multiple punctate fluorescein leakage and increased capillary permeability at 31 months formed a larger nonperfusion area (asterisk) at 33 months. B, Fluorescein angiogram at 35 months of feeding. Blockage of background fluorescence that suggested the occluded area was seen in some areas (asterisk). Several capillary closures with the obstruction of arterioles could be observed (arrow). C, Obstruction of the main retinal vessels, sausagelike dilation, and serpiginous veins (arrow) at 39 months of galactose feeding.
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Figure 3. Color fundus photograph of the dog retina from the galactose-fed group after 35 months of feeding. Spotted retinal hemorrhages can be seen.
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In fluorescein angiograms obtained after 41 months of galactose feeding, 7 (78%) of 9 dogs developed areas of nonperfusion (Figure 4). The numbers in these fluorescein angiograms correspond to the individual dog numbers. In the 2 galactose-fed dogs not demonstrating areas of nonperfusion, some spots of fluorescein leakage were observed in one, while the other dog had no abnormal retinal findings. The incidence of retinal lesions in the galactose-fed group at 30, 37, and 41 months of feeding is summarized in Table 2. No abnormal findings, such as hemorrhages or exudate, were observed in the control group.
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Figure 4. Panorama fluorescein angiograms obtained after 41 months of galactose feeding. Numbers in these fluorescein angiograms correspond to individual dog numbers. The retina of one dog showed only a few leakage spots at the nasal upper retinal side, while the other dog had no abnormal retinal findings.
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Table 2. Incidence of Retinal Lesions in the Galactose-Fed Group (n = 9)
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COMMENT
The occurrence of retinal lesions similar to those seen in human diabetic retinopathy has been reported in dogs made galactosemic by the long-term administration of a galactose diet and dogs made diabetic by the administration of alloxan.1-3,8 Moreover, the appearance of lesions associated with proliferative retinopathy in galactose-fed dogs indicates that the galactose-fed dog is a suitable animal model for investigating human diabetic retinopathy.3 Previous reports1-6,8-10 on the progression of retinal changes in galactose-fed dogs have focused on histological observations that retinal changes progress from the initial destruction of pericytes to endothelial cell proliferation and the formation of microaneurysms and their subsequent degeneration, the appearance of hemorrhages and cotton-wool spots, vessel occlusion and the formation of areas of nonperfusion, intraretinal microvascular abnormalities, increased hemorrhages, and new vessel growth into the vitreous. Because cataract formation occurs rapidly in galactose-fed animals, fundus examinations are difficult to conduct in dogs after several months of galactose feeding.7 By extracting the lens prior to galactose feeding, regular fluorescein angiography and fundus color photography could be conducted throughout the entire study (41 months) in dogs fed a 30% galactose diet.
Clinical lesions in human background diabetic retinopathy include microaneurysm formation, retinal hemorrhages, the formation of hard exudate, and retinal edema.11-12 In the present study, retinal lesions observed in the aphakic galactose-fed dogs include the formation of microaneurysms, retinal hemorrhages, capillary networks demonstrating fluorescein leakage, and retinal nonperfusion. Dilated microvascular lesions demonstrating fluorescein leakage, which resemble intraretinal microvascular abnormalities in human preproliferative retinopathy, were also observed. These clinical observations correlate well with the previous histological reports of retinal lesions in galactose-fed dogs.
In the progression of retinal changes observed by fluorescein angiography, leakage of small spotty fluorescein dye at the posterior pole along the temporal quadrant was first observed after 28 months of galactose feeding. Although this leakage stopped within 2 months after its onset, capillary obstruction, as indicated by the appearance of small areas of fluorescein nonperfusion, developed at the same area previously demonstrating the fluorescein leakage. Subsequent development of multiple fluorescein-leaking spots surrounding the small area of nonperfusion was observed 1 month later (after 31 months of galactose feeding). As leakage stopped, the area of fluorescein leakage formed into an area of nonperfusion devoid of blood supply. This process was repeated and a broad retinal area of nonperfusion was formed by the confluence of small nonperfused areas. Results from repeated fundus examinations at the same loci provided information on how retinal lesions such as fluorescein leakage and areas of nonperfusion develop and change. The appearance of microaneurysms, detected as a small leaking points of dye by fluorescein angiography, disappear as the disease progresses. This disappearance is not due to the disappearance of the microaneurysm structure itself, but to the occlusion of the microaneurysms' lumen by the aggregated blood elements or hyalinization and atrophy of the aneurysm and/or afferent vessels, which results in the hypoperfusion of the contrast medium (fluorescein dye) and the cessation of fluorescein leakage. These changes in the progression of galactose-induced retinopathy in dogs appear identical to the development of human diabetic retinopathy. This strongly indicates that retinal changes occurring in the galactose-fed dog model can be traced to the initial-stage lesions in human diabetic retinopathy described by Cogan et al13-15 more than 30 years ago and Kador16 in 1988. These lesions begin at the temporal side of the retina and expand to the nasal side. During the maximum observation period of 41 months in our study, a considerable variation in the severity of retinopathy in the galactose-fed dogs was observed. In the most advanced cases, virtually all of the upper half of the retina became nonperfused with the presence of intraretinal microvascular abnormalities, while no fluorescein leakage was observed in the retina of 1 galactose-fed dog. The cause of this variation is not clear at this time because all galactose-fed dogs had similar blood galactitol levels. It should be emphasized that, to our knowledge, there have been no reports indicating the appearance of clinical signs such as hemorrhage or fluorescein leakage in rodent animal models.
AUTHOR INFORMATION
Accepted for publication March 3, 1998.
We thank Peter F. Kador, PhD, for providing helpful discussions.
Reprints: Yoshio Akagi, MD, PhD, Department of Ophthalmology, Fukui Medical University, 23 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-11, Japan (e-mail: akagiy{at}fmsrsa.fukui-med.ac.jp).
From the Department of Ophthalmology, Fukui Medical University (Drs Kobayashi, Kubo, Takahashi, and Akagi) and Fukui Institute for Safety Research, Ono Pharmaceutical Co Ltd (Drs Kasahara and Yonezawa), Fukui, Japan.
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