The Patency of the Retinal Vasculature to Erythrocytes in Retinal Vascular Disease

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1 Investigative Ophthalmology & Visual Science, Vol. 31, No. 3, March 1990 Copyright Association for Research in Vision and Ophthalmology The Patency of the Retinal Vasculature to Erythrocytes in Retinal Vascular Disease Joshua Den-Nun,*t Valerie A. Alder,f Ian J. Consrable,t and Claire E. Roberfsf This paper presents the first evidence that in retinas with experimentally induced vascular disease some vessels contain only plasma. This was demonstrated by a histologic technique developed specifically to test the hypothesis that at some stage in retinal vascular disease, vessel patency to erythrocytes is lost before vessels close to plasma. Using this technique, we visualized three major components of the circulation at all retinal locations: the erythrocytes; the plasma as marked by the presence of 0.2-Mm fluorescent microspheres; and all functioning endothelial cell nuclei, which were marked by the fluorochrome bis-benzimide. It was assumed that the distributions of the erythrocytes and small particles in retinal whole mounts reflected accurately the true in vivo distributions at the moment of circulation arrest. Postenucleation the retina can be viewed and photographed within 45 min of circulation arrest. The technique was used on normal rats and on rats induced with a fast-developing model of retinal vasculopathy. With this model, we demonstrated retinal vascular segments perfused by plasma but containing no erythrocytes with functioning endothelial cells in the vessel walls. This may mean that an early factor in some retinal vascular pathologies is tissue hypoxia caused by reduced erythrocyte perfusion. Invest Ophthalmol Vis Sci 31: ,1990 Alterations in structure and function to the retinal microvasculature are intimately associated with the pathogenesis of some retinopathies that have a local or systemic origin. Some of these retinopathies, such as diabetic retinopathy, are major causes of blindness or visual loss. Although the initiating factors and early stages in these various retinopathies may differ, it is widely accepted that the common final pathway is impairment to the blood supply, especially with regard to oxygen transport and delivery. One fundamental question is whether progressive retinal ischemia caused by the narrowing and ultimate occlusion of the retinal microcirculation plays a key role in the early stages of the pathology, or whether ischemia exerts an effect only in the later stages. A demonstration that reduced erythrocyte perfusion plays an early role in vascular pathology would be of great importance, not only for our un- From the *Department of Ophthalmology, Tel Aviv Medical Centre, Tel Aviv, Israel, and the I Lions Eye Institute and Department of Surgery, University of Western Australia, Nedlands, Australia. Supported by the Juvenile Diabetes Foundation International, the National Health & Medical Research Council and the Lions Save Sight Foundation. JB-N held a Shaw Foundation Fellowship from the QE2 Medical Centre, Perth. Submitted for publication: February 8, 1989; accepted June 21, Reprint requests: Dr. V. A. Alder, Lions Eye Institute and Department of Surgery, University of Western Australia, Nedlands, WA 6009, Australia. derstanding of the disease, but also for treatment regimens. In this study, we explored the hypothesis that in the early stages of a retinal micro vasculopathy, areas of the microvasculature lose their patency to erythrocytes while still maintaining patency to plasma. Loss of patency to erythrocytes could be the direct consequence of vessel wall and lumen changes or of a reduction in erythrocyte deformability. If there is a difference in the relative patency to plasma and erythrocytes, hypoxia may be vitally important in the incipient stages of the development of these disorders. To address this question, we developed a new histologic technique which we refer to as the vascular trichrome technique. We assume that this technique has the property of allowing the simultaneous visualization of three important functional components of the retinal vasculature as they are at the time of circulation arrest. These three components are the functioning endothelial cells stained by the lathogen bis-benzimide, 1 the erythrocytes, and 0.2-/i,m microspheres, which represent the plasma moiety of blood. As an initial test of the hypothesis of relative vascular patency and also as an evaluation of the power of our histologic technique to relative patency, we used a rat model of fast-developing chemically induced retinal vasculopathy. Currently we are testing the same hypothesis in other animals inflicted with retinal microvascular disease that bears a closer analogy to human retinal microvascular pathology. 464

No. 0 REDUCED PATENCY OF RETINAL VA5CULATURE TO ERYTHROCYTES / Den-Nun er ol 465 We sought to confirm our hypothesis by demonstrating that early in the disease when the elements of the vessel walls

2 No. 0 REDUCED PATENCY OF RETINAL VA5CULATURE TO ERYTHROCYTES / Den-Nun er ol 465 We sought to confirm our hypothesis by demonstrating that early in the disease when the elements of the vessel walls (the endothelial cells and pericytes) appear still to be functioning there are vascular segments that are not patent to erythrocytes but that still pass plasma. Indeed, this would imply that the loss of patency to erythrocytes can occur before the capillaries become acellular. Materials and Methods Vascular Trichrome Technique The technique was developed to demonstrate three major functional components of the microcirculation at the same retinal location: the erythrocytes, fluorescent microspheres distributed uniformly in plasma, and endothelial cells. The rat was anasthetized by spontaneous respiration of Halothane (3%; ICI, Australia) through a mask. The femoral vein was cannulated, and 250 U (1000 U/ml) of Heparin sulphate (David Bull Mulgrave, Victoria, Australia) was injected. The lathogen 2'-(4-ethoxyphenyl)-5-(4-methyl-1 -piperazinyl)-2,5'- bi-lh-benzimidazole trihydrochloride trihydrate (bis-benzimide, Hoechst 33342; Sigma, St. Louis, MO) was made up to a solution of 5 mg/ml in 0.9% NaCl and was perfused through the vein over 30 sec to result in a total dose of 10 mg/kg body weight. Fluorescent polystyrene microspheres (Duke Scientific, Palo Alto, CA), 0.2 /xm in diameter, were perfused at 0.2 ml/min for 5 min. Loops were placed around the optic nerves of both eyes, and both ocular circulations were clamped abruptly to cause instant arterial occlusion. This technique was not traumatic to the retina, and blood flow was assumed to be arrested instantly, such that the components of the blood remained in the same location as they were at the time of occlusion. The eyes were enucleated and placed in 10% buffered formol-saline solution (ph 7); immediately afterwards, the animal was sacrificed by barbiturate overdose. Fifteen minutes after enucleation, the anterior segments and lenses were removed, and the retinas were washed directly with a gentle flow of 10% buffered formol-saline while being dissected free from the underlying choroid. They then were wholemounted and coverslipped. Within 20 min after enucleation, the retinas could be observed with normal and fluorescence microscopy. The erythrocytes were observed under normal microscopy, since the brown hemoglobin can be visualized easily. The 0.2-/im microspheres were observed under fluorescence microscopy (excitation filter 470 nm, barrier filter 530 nm) and appeared green. The endothelial cells were observed under fluorescence microscopy (exitation filter 360 nm, barrier filter 455 nm) and appeared blue. Any one retinal area could be observed and photographed sequentially with the appropriate illumination to reveal each of the three vascular components in turn, thus producing three views of the same retinal area at any depth. The whole procedure, from initial bis-benzimide injection to retinal photography, could be performed easily within 45 min. Since the bis-benzamide is not specific for endothelial cell nuclei, it gradually diffused to cells farther from the vessel lumen. Some time later, pericyte nuclei could be observed; finally, after several hours, the nuclei of all retinal cells were stained, giving the retina a diffuse blue fluorescence. The retina was in a good state for photography of endothelial cells and pericytes for 2-3 hr. The deterioration of the quality of the bis-benzimide interaction with the endothelial cell nuclei did not affect the clear visualization of the other two components. Acute Retinopathy Model A fast-developing vasculopathy model (Heath and Rutter 2 ) was induced in rats to test the power of the vascular trichrome technique. Animals used were 40 albino male Wistar rats (10-12 weeks old, kg body weight). Diabetes was induced by a subcutaneous injection of Alloxan (Sigma) in a 2% phosphate-citrate-buffered solution, ph 4.5, 3 (200 mg/kg body weight). After 4 days, a combination of polydipsia, polyuria, and hyperglycemia confirmed the development of diabetes mellitus. At this stage, imino-dipropionitrile (IDPN; Allwest Scientific, Perth, W. Australia) in a 20% aqueous solution was given subcutaneously (1 g/kg body weight). Starting at the end of the 1st week after the IDPN injection, groups of rats were sacrificed at various time intervals after being subjected to the vascular trichrome technique. No animal was kept for longer than 4 weeks after the IDPN injection. All investigations in this study conformed to the ARVO Resolution on the Use of Animals in Research. Normal Retina Results For each retinal area three photographs were produced. Figures 1A and 2A show the erythrocyte perfusion of the inner and outer vascular bed, respectively, for the same retinal area; the only difference between the two views is the focal plane of the microscope. These were viewed under low power. The fluo-

3 466 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1990 Vol. 31 Fig. 1 (Left). Superficial retinal vascular bed of the normal rat retina (A [top]) Erythrocytes. (B [center]) Fluorescent microspheres. (C [bottom]) Endothelial cell nuclei stained with bis-benzimide. (XI50) Fig. 2 (Right). Deeper retinal vascular bed of the same area shown in Figure 1. (A [top]) Erythrocytes. (B [center]) Fluorescent microspheres. (C [bottom]) Endothelial cell nuclei stained with bis-benzimide. (X1S0) rescent microspheres representing plasma perfusion for the same two beds at the same location are shown in Figures IB and 2B. Figures 1C and 2C represent the bis-benzimide-stained endothelial cell nuclei at the same two focal planes of the retinal whole mount. Figures 3A-C show a smaller retinal region under higher power to demonstrate the detail and information available with the vascular trichrome technique. In Figure 3A, eight individual erythrocytes are visible in one capillary; the bis-benzimide-stained endothelial cells at the same location are depicted in Figure 3B. The more weakly fluorescing cell nucleus in the center probably belongs to a pericyte. Pericytes fluoresced maximally after the endothelial cells started to lose their bis-benzimide stain. Figure 3C shows the same area with both normal and fluorescence illumination to demonstrate the relationship between the erythrocytes and the endothelial cells and pericyte.

No. 3 REDUCED PATENCY OF RETINAL VASCULATURE TO ERYTHROCYTES / Den-Nun er ol 467 Fig. 3 (Left). High magnification of one normal retinal capillary of the deeper capillary bed.

4 No. 3 REDUCED PATENCY OF RETINAL VASCULATURE TO ERYTHROCYTES / Den-Nun er ol 467 Fig. 3 (Left). High magnification of one normal retinal capillary of the deeper capillary bed. (A [top]) Individual erythrocytes. (B [center]) Three bis-benzimide-stained endothelial cell nuclei together with one pericyte nucleus. (C [bottom]) Composite photograph showing relative locations of the erythrocytes, the endothelial cell nuclei, and the pericyte nucleus. (X750) Fig. 4 (Right, top). Bis-Benzimide stain of an arteriovenous crossing in rat retina demonstrating the clear distinction between the circular boundary of the venous endothelial cell nuclei and the elongated endothelial cell nuclei of the artery. (X200) Fig. 5 (Right, center and bottom). Retinal vascular bed of an alloxan + IDPN rat. (A [center]) Erythrocytes. (B [bottom]) Fluorescent microspheres. These demonstrate that erythrocyte perfusion over large areas of the vascular bed is absent, whereas there still is perfusion by thefluorescentmicrospheres. (X200) The arteriovenous crossing shown in Figure 4 illustrates the rounded endothelial cell nuclei of the veins compared with the more elongated nuclei of the arteries and capillaries. This difference facilitated the identification of arteries and veins. Acute Retinopathy Model The effect of the dual dose of alloxan and IDPN on the retinal vasculature was rapid and thorough. The retinas appeared grossly abnormal within 10 days

468 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / March 1990 Vol. 31 Fig. 6. Retinal vascular bed of an alloxan + IDPN rat. (A [lop]) Erythrocyte perfusion. (B [center]) Microsphere perfusion.

5 468 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / March 1990 Vol. 31 Fig. 6. Retinal vascular bed of an alloxan + IDPN rat. (A [lop]) Erythrocyte perfusion. (B [center]) Microsphere perfusion. (C [bottom]) Endothelial cell nuclei. The side vessel is not perfused by erythrocytes, whereas microsphere perfusion is evident and the endothelial cells are present in this side vessel. (X200)

No. 3 REDUCED PATENCY OF RETINAL VA5CULATURE TO ERYTHROCYTES / Den-Nun er ol 469 after the injection of IDPN. Large areas were totally lacking in erythrocytes (Fig. 5A).

6 No. 3 REDUCED PATENCY OF RETINAL VA5CULATURE TO ERYTHROCYTES / Den-Nun er ol 469 after the injection of IDPN. Large areas were totally lacking in erythrocytes (Fig. 5A). When the same area was photographed to visualize the fluorescent microspheres, it was clear that the vessels in this area were still patent to the microspheres (Fig. 5B). However, the vascular bed was tortuous with the sudden changes in vessel diameter. In the early stages, the arterial side of the circulation, in the innermost capillary bed and close to the disk, was affected, with the vessels in the retinal periphery having a normal appearance. The size of the damaged region rapidly spread, however, so that by 3 weeks after the IDPN injection, all of the vessels of the retina showed similar pathology. Vessel segments in which no erythrocytes were present but which were well perfused by microspheres were very numerous. Figure 6 demonstrates that erythrocytes were present in the main vessel but absent in the side vessel (Fig. 6A); that the microspheres were present in both the main and side branches (Fig. 6B); and that the endothelial cells were stained by the bis-benzimide, indicating that they still were functional (Fig. 6C). Usually the regions showing no erythrocyte perfusion had labeled endothelial cells. In regions where vessel lumen was occluded, the vessel wall appeared swollen. Frequently, erythrocyte perfusion appeared to stop at an arteriole branch, with evidence of erythrocyte blockage; the following arteriole, however, certainly was perfused by microspheres and therefore plasma. This point is substantiated in Figure 7, which shows that the erythrocytes were blocked at the junction of the vessels (Fig. 7 A), whereas microspheres were present in the distorted side vessel where it leaves the plane of the photograph (Fig. 7B). The same situation is apparent in Figure 8, which demonstrates a main vessel well perfused by erythrocytes, with a lone erythrocyte at the junction of the branch vessel which was otherwise totally unperfused by erythrocytes (Fig. 8A). However, there still was evidence of microsphere perfusion and hence plasma perfusion in the narrowed side branch (Fig. 8B) where it leaves the plane of focus of the photograph. Fig. 7 {Left). High magnification of an arterial branch of an alloxan + IDPN retina. (A [lop]) There is blockage to erythrocyte perfusion, while microsphere perfusion still is apparent (B [bottom]). (X750) Fig. 8 (Right). High magnification of an arterial branch of an alloxan + IDPN retina. {A [top]) There is blockage of a side vessel to erythrocyte flow, resulting in swollen walls, but the vessel is patent to fluorescent microspheres (B [bottom]). (X750)

7 470 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1990 Vol. 31 Discussion These experiments demonstrated a difference in the retinal whole-mount distributions of plasma and erythrocytes in normal retinas and in retinas with a fast-induced vascular pathology. In normal retinas, the retinal whole mounts never showed vessel segments completely devoid of erythrocytes. Erythrocytes, plasma, and functioning endothelial cells and pericytes could be visualized in every vessel segment, in both capillary beds and at all retinal locations. In contrast, in the pathologic retinas, there were vessel segments in which no erythrocytes could be visualized, while the vessel lumen still contained plasma and the endothelial mural cells of the vessel still were functioning, insofar as they took up bis-benzimide stain. Despite all of the precautions we took to ensure that the distributions of erythrocytes and microspheres reflected their in vivo dynamic distributions, we cannot totally rule out the possibility that the procedure itself affected the erythrocyte distribution. If it does, however, the effect in normal retina is very different from that in the retinas with the experimentally induced vascular pathology. The presumed consequence of an altered distribution of erythrocytes in retinas with early micro vascular disease is concomitant tissue hypoxia in regions supplied by vessels which are either totally or partially blocked to erythrocyte flow. We currently are applying the vascular trichrome technique to ascertain whether loss of patency to erythrocytes can be demonstrated in animal models that are more relevant to human retinal vascular disease, such as the more slowly developing retinopathies of the streptozotocin-induced or the galactosemic rat models of diabetic retinopathy. A large group of retinal vasculopathies with a variety of different origins have a similar end point vascular damage resulting in capillary occlusion and nonperfusion, as demonstrated by fluorescein angiography. The common denominator among these different disorders may be a tissue ischemia that develops during the progress of the disease, due to oxygen transport and delivery problems. If the relative perfusion of erythrocytes and plasma (microspheres) that we observed in vessels of the IDPN + alloxan rats can also be demonstrated in acceptable animal models of human retinal vascular disease, then we may have uncovered the common pathway whereby vascular diseases with different origins can progress to the same end point. The main advantage of the vascular trichrome technique is that the relative distributions of microspheres and erythrocytes are dictated by the actual flow in the animals' vessels at the time of circulation arrest. With the bis-benzimide, one can determine the regions with functioning endothelial cells and can observe the endothelial cells' relationship to the erythrocyte and plasma flow. Furthermore, the ease and speed of the retinal processing serves to decrease tissue damage and possible artifacts. We experienced no difficulty with viewing the whole retina in three dimensions, and both capillary beds could be separated easily, as is shown in Figures 1 and 2. The trichrome technique also allows early observation of the retina after enucleation, after which the investigator may choose the areas of interest for more detailed study by electron microscopy and conventional light microscopy. In these respects, the role of the vascular trichrome technique in retinal vascular histology may be complementary to the widely used technique of trypsin digest and PAS staining. 4 We believe that this method allows important insights into the possible physiologic changes that occur in retinal vascular disease. Key words: retinal vascular disease, erythrocyte perfusion, alloxan, imino-dipropionitrile, rat Acknowledgments The authors wish to acknowledge the expert technical assistance given by Mr. Michael Brown and Mr. Peter Burrows. References 1. Reinhold HS and Visser JWM: In vivo fluorescence of endothelial cell nuclei stained with the dye Bis-benzimide H Int J Microcirc Clin Exp 2:143, Heath H and Rutter AC: Retinal angiopathy in the imino-dipropionitrile-treated alloxan-diabetic rat. Br J Exp Pathol 67:116, Klebanoff SJ and Greenbaum AL: The effect of ph on the diabetogenic action of alloxan. J Endocrinol 11:314, Kuwabara T, and Cogan DG: Studies of retinal vascular patterns: I. Normal architecture. Arch Ophthalmol 64:904, 1960.

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