I n the past two decades a number of

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1 Development of retinal vessels in the rat Paul Henkind and Luiz F. De Oliveira* With the technical assistance of Linda Karasik The development of retinal vessels in the rat from birth to 5 days of age is described. Various techniques including India ink injections into the vascular system, retinal digestion, PAS stained whole mounts, and combinations of these methods were utilized. The first stage in retinal vascularization is the ingrowth of an undifferentiated mesh or syncytium of mesenchymal cells into the peripapillary retina. Later a primitive capillary net forms from the initial meshwork. In a third stage larger vessels, i.e., early arterioles and venules, make their appearance, developing out of the capillary network and possibly from the syncytium directly. The capillary-free zone around arterioles is a late development in vasculogenesis and occurs by retraction of mesenchymal strands and primitive capillaries from around the developing arteriole. I n the past two decades a number of papers have been written about the growth and development of retinal vessels. Several investigators including Michaelson, 1 ''- Ashton, 8-4 and Cogan n have worked and made contributions in specific areas of the subject. Nonetheless, it is difficult to detect any uniformity of opinion as to how retinal vessels develop, particularly in the early stages of retinal vascularization. Michaelson, 1 ' - studied specimens from several animal species which were injected with India ink, as well as blood-gelatin injected material from man. From these investigations he was convinced that retinal capillaries bud from venules which grow into the retina from the head of the optic From the Department of Ophthalmology, New York University School of Medicine, New York, N. Y. This work was supported by Grant No. NB , National Institute of Neurological Diseases and Blindness, National Institutes of Health. "NATO Fellow (Portugal). 520 nerve. Cogan 5 examined digest preparations of human fetal retinas and concluded that solid cords of endothelial cells bud from the hyaloid system of vessels at the head of the optic nerve and enter the retina; later a chicken-wire plexus of primitive capillaries develop from these cords. Ashton 3> 4 with regular cross sections, flat mounts, and digest preparations pointed out that one could find undifferentiated spindle cells preceding vascular budding; these cells are associated with PAS-positive glycogen granules, 0 and stain positively for alkaline phosphatase activity. 7 He further stated that in human fetuses the vanguard of the invading growth consists of mesenchymal precursors and not of solid cords of endothelial cells nor of fully formed capillary buds. 1 However, in the cat and rabbit he felt that normal retinal vascularization occurred by a process of budding without mesenchymal precursors being present. Recently, Engerman and Meyer, s using a special PAS technique to study whole mounted rat retinas, found that primitive

2 Volume 6 Number 5 Retinal vessel development 521 capillaries extended between and anterior to the tips of larger radial vessels. Agrawal using the digestion method to study the developing retinal vessels in the rat noted that the peripheral portion of the retina shows proliferative cells which are undifferentiated and do not show any capillary network. We have re-examined the development of the retinal vascular bed of the rat. A variety of techniques including PAS stained whole mounts, India ink injection studies, digest preparations, and a combination of these methods were used in this work. Our findings are described, and their relationship to previous concepts is discussed. Material and methods Stock albino rats were used in this work. Experiment 1. Thirty-five newborn rats ranging in age from several hours to 5 clays were anesthetized with ether, their chests were opened, and in each animal the left ventricle was punctured with a thin plastic tube attached to a syringe containing several milliliters of 0.2 per cent sodium nitrite. The right auricle was incised and sodium nitrite injected until the fluid flowing from the auricle was clear. The syringe was then replaced with one containing stock India ink diluted in half with normal saline. The ink was injected manually until the head and upper extremities of the rat were black. The eyes were enucleated and placed in Heidenhain's Susa fluid; afterward the retinas were dissected free, mounted flat, and stained with periodic acid-schiffs reagent according to the technique of Engerman and co-workers. 10 The preparations were examined by transmitted light microscopy with the use of a green filter (Wratten No. 58) to improve visualization of the vessels. In a number of instances one eye was removed before the ink injections took place, but it was otherwise processed as above. Experiment 2. The retinas from 17 rats, which were several hours to 5 days old, as well as a few full-term fetuses, were digested by the pepsintrypsin method, 11 using a shortened digestion period; i.e., never more than 5 minutes in either solution. The pepsin and trypsin solutions were warmed to 36 C. before digestion was started. The mounted retinas were stained with PAS and hematoxylin or alternatively PAS with hematoxylin and eosin and examined by light microscopy. A number of rats were injected with India ink prior to having the eyes removed and the retinas digested. In addition, several 5-day-old rats had a ligature gently placed around the optic nerve of one eye in an attempt to block venous outflow and yet permit arteriolar inflow, with the resultant vascular congestion of the retina. Results and observations. The various experimental techniques complimented each other. Whole mounts stained with PAS allowed us to see the developing vascular bed in situ. Avascular cordlike extensions were seen to extend beyond the area of patent (inked) vessels. Retraction of the vascular strands around developing arterioles could also be studied. Digest preparations gave a better idea of the entire vascular complex including the leading edge consisting of a mesh or syncytium of undifferentiated cells. This latter technique also permitted detailed study of the cellular morphology of the various components of the vascular complex. During the first week after birth, all of the retinal vessels of the newborn rat lie in a two dimensional plane near the inner surface of the retina; later a deep capillary network appears. This is similar to the observations of Michaelson, 2 Ashton and Blach/ 2 and Engerman and Meyer. s In the rat one can distinguish several stages in early retinal vascular development. The first stage appears to consist of an ingrowth of an undifferentiated cellular meshwork, or syncytium, into the peripapillary retina, and this is visible only in a digest preparation of a full-term fetus (Fig. 1) and not in PAS stained whole mounts. This meshwork appears to be a direct extension of tissue derived from the vessels of the optic nerve head, and it seems to be composed of primitive mesenchymal cells. Shortly thereafter, a network of highly cellular vessels of small caliber develops within the proximal portion of this meshwork (Fig. 2). These young vessels surround oval or pentagonal spaces which initially contain processes and nuclei from the primitive vascular mesh. Some of the pathways are patent to India ink, while others are not (Fig. 3), and may indeed be solid cords. At this second stage, which lasts for a day or more, it is impossible to differentiate arterioles and

3 522 Henkind and De Oliveira Investigative Oph thahnologxj October 1967 Fig. 2. Incomplete retinal digest from a rat less than 24 hours old. Vascular cords, some containing blood, can be seen extending from the disc into the undigested neural tissue. (PAS, hematoxylin and eosin. Original magnification x!60.) Fig. 1. Incomplete retinal digest preparation from a full-term fetal rat. Strands (S) of undifferentiated mesenchyme can be visualized around the disc, and spindle-shaped nuclei (N) of the cells forming the vascular meshwork are present elsewhere. (PAS, hematoxylin and eosin. Original magnification x250.) venules. Later, in a third stage, one can delineate primitive arterioles and venules, which appear as larger, more well-developed channels forming arcades within the vascular network (Fig. 4). The process of maturation takes place from the disc outward, and the periphery of the vascular complex is always more primitive in appearance than the central area (Fig. 5). Four to 5 days after birth this primitive meshwork is present only at the periphery of the growing vascular tree (Fig. 6). Vessel inking does not extend evenly out from the disc to include all the primitive capillaries. Indeed one may see more ink in the middle of the bed than around the disc. The cells composing the initial vascular meshwork have prominent nuclei, which are elongated and irregular, and may be spindle-shaped, sickle-shaped, triangular, or rectangular in appearance. Mitotic figures were common (Fig. 7), but the mitotic index was not calculated. The cytoplasm was pale, scant, and filamentous and seemed to stream out of the end of the cell and intercommunicate with strands from adjoining cells; the whole appearance is of a distorted net with enmeshed cells. This meshwork is fragile and easily lost unless great care is taken during the process of digestion (Fig. 8). Highly cellular, thick cords, which are larger than those of the early capillary

4 A A. Fig. 3. Whole mount of a 2-day-old rat retina inked and stained with PAS. (A) Photographed with ordinary transmitted light. (B) Same preparation photographed with use of a Wratten No. 58 filter. Note the cords continuous with but extending beyond the inked channels. (Original magnification x320.) ' Fig. 4. Retinal digest from a 3-day-old rat. In this preparation we see the undifferentiated mesenchymal meshwork at the periphery, the advancing zone of cords, the primitive capillary meshwork, and a larger vascular arcade (arrow). At this stage it is impossible to delineate a capillary-free zone around the larger vessels. (PAS and hematoxylin. Original magnification x87.)

5 524 Henkind and De Oliveira In Destigatice Ophthalmology October 1967 Fig. 5. Retinal digest from a 5-day-old rat (ligature around optic nerve). As in Fig. 4 the three components of the vascular complex are easily seen, as well as vascular arcades. In addition a capillary-free zone is beginning to become evident around the primitive arterioles, (PAS, hematoxylin and eosin. Original magnification x50.) Fig. 6. Digest preparation from a 5-day-old rat. Vascular cords and aligned cells are seen in the peripheral cellular meshwork. Most are continuous with patent vessels. (PAS, hematoxylin and eosin. Original magnification xloo.)

6 Volume 6 Number 5 Retinal vessel development 525 Fig. 7. Mitotic stages in cells from different zones of the developing retinal vascular complex of the rat; (1) prophase, (2) metaphase, (3) early anaphase, (4) anaphase, (5) telophase, (6) telophase (white arrow) and prophase (black arrow). Digest preparations. (PAS and hematoxylin. Nos. 1, 2, 3, 5, and 6, original magnification xl,000; No. 4, original magnification xl,200.) Fig. 8. Digest from a 4-day-old rat. Note loss of the mesenchymal meshwork at the upper left and right sides of the preparation. This gives the impression that the complex ends as cords in these areas, but the upper center of the picture shows that this is not really so. (PAS and hematoxylin. Original magnification x45.)

7 526 Henkind and De Oliveira Investigative Ophthalmology October 1967 mesh, are occasionally seen within the mesenchymal network before the primitive capillary bed has developed in the area. These channels which may become arterioles or venules seem to be without major connections to patent channels (Fig. 9). On the other hand, most of the larger early vessels destined to be arterioles or venules seem to develop from the initial capillary network, and they are arranged in arcades extending from the disc out to the beginning of the cellular meshwork (Fig. 10). The perivascular capillary-free zone appears to arise as a result of atrophy and Fig. 9. Digest from a 5-day-old rat (ligature around optic nerve). At the right hand side of the picture vascular cords (arrow) with little or no attachment to the patent, more centrally placed vessels are seen. (PAS, hematoxylin and eosin. Original magnification *200.) t. \ >. #m33 py Fig. 10. India inked and digested retina from a 1-day-old rat. Note the degree of inking compared to the full extent of the vascular complex. The most prominent channels are arranged as arcades. (PAS, hematoxylin and eosin. Original magnification xl25.)

8 Volume 6 Number 5 Retinal vessel development 527 retraction of primitive channels around the developing arterioles (Fig. 11). No such zone can be seen in the early stage when whole mounts are inked and stained with PAS and examined through a green filter. Discussion It is now widely accepted that the embryonic vascular system in general arises from mesenchymal tissue at various sites within the body. 13 ' 11 It is also agreed that once the embryonic circulation is fully established, the extension of the vascular system is solely by growth from pre-existing vessels and not from isolated portions of mesenchyme. 14 Where does the developing retinal vascular system fit into the previously mentioned scheme? It develops rather late in embryonic or even in postembryonic life after most of the systemic vascular system has been developed. Since the avascular retina lacks mesenchymal elements, 2 vessels must develop from tissues entering the retina from without, and then logically from an extension of a functioning vascular system. Indeed there seems no disagreement over the point that retinal vessels develop from ingrowing tissue rather than Fig. 11. Whole mount of an inked PAS stained 5-day-old rat retina. (A) A zone completely free of vascular components is seen around a retinal arteriole. Transmitted light. (B) Same as (A) but photographed with a Wratten No. 58 filter. Note the numerous vascular strands in the capillary-free zone. {Original magnification x800.)

9 528 Henkind and De Oliveira Investigative Ophthalmology October 1967 from some intraretinal component. As pointed out in the introduction there is controversy as to exactly what components enter the retina and in what form to initiate retinal vascular development. Michaelson 2 mentions first that solid buds grow from the vessels of the optic nerve, enter the retina, and apparently differentiate into venules and arterioles which later form capillaries. This point of view has few adherents at present, and indeed the weight of evidence is against it. 4 ' F> ' s Our work indicates that a rather undifferentiated meshwork of cells, presumably mesenchyme, extends from the vessels in the optic nerve and invades the innermost layer of the peripapillary retina. Mitotic activity is pronounced among these cells within the retina. This meshwork which is to be found at the leading edge of the developing vascular complex seems to be similar to the undifferentiated spindle cells, thought to be mesenchymal elements, which were demonstrated in human fetal retinas by Ashton, 3-4 Serpell/' 1 and Nilhausen. 7 The contiguity of these cells with the more definitive though immature retinal vessels is well-demonstrated in the digest preparations (see Ashton's Fig. 2 4 ). If extreme care is not taken during the digestion procedure, this meshwork will be lost, and the impression will be that either the solid cords or a capillary plexus are the vanguard of the vascular complex. Similarly, Cogan 5 showed solid cords of endothelium in digest preparations of human embryos, which he considered to be the most advanced edge of the early retinal vasculature. It appeared in the rat that these cords were only a portion of a more extensive plexus (Fig. 8). One point at least seems clear; it is difficult to be certain that the ingrowing cords are composed of endothelium as stated by Cogan. 5 More likely they are immature mesenchymal elements which will later differentiate into endothelial cells and intramural pericytes. 15 We have used the term vascular complex in regard to the developing retinal vascular bed of the rat, and we wish to clarify its meaning. Various stages of vascular development are found in the rat at birth to several weeks after birth (here we have concentrated on the first 5 days of life). Initially, one sees only an interconnected meshwork, perhaps a syncytium of primordial elements without any hint of vascular channels. This meshwork is also represented in the peripheral portion of the more welldeveloped vascular system seen on days 2 to 5. Just central to the undifferentiated mesh is a network of primitive capillaries, some patent, some apparently not, and little or no evidence of arterioles or venules.* Extending centripetally back to the disc is a more developed though still immature vascular pattern with a more well-defined capillary bed and early arterioles and venules. This entire complex can be digested from the retina in toto and is a part of the developing vascular tree of the retina. Maturation takes place centrifugally from the disc, but in some places isolated a vascular strands, some of even larger caliber than capillaries, seem to be present in the primordial syncytium. This appears to be further evidence of the potential of this tissue to form more definitive vessels without direct budding from a formed vascular tree. Indeed the initial syncytium is the basis for vascular development rather than just a support which other elements use. This may be an important point in vascular embryogenesis elsewhere in the body, but it may be difficult to prove since digest preparations have not been feasible except with retinal or brain tissue. 30 It seems certain that the earliest retinal arterioles and venules develop from the immature capillary bed and not vice versa. There is also evidence that arterioles and venules may develop from the mature retinal capillary bed. 17 These findings are consistent with observations made on developing vessels in other organs. They 1Si ]i) do "While the terms capillary, arteriole, and venule are used to describe the early retinal vasculature, these vessels do not morphologically resemble those in adult life, but in time will evolve into the mature forms.

10 Volume 6 Number 5 Retinal vessel development 529 not imply, however, that new capillaries cannot bud out from formed retinal venules, for this is possible in a variety of ocular disorders. To this point in the discussion we have been concerned with the appearance of the various components of the retinal vascular bed and have not discussed the factors leading to vascularization. These factors are still in the realm of speculation and have been discussed by Michaelson, 2 Ashton/ 0 and in general terms by Arey. 14 One last point for discussion concerns the capillary-free zone found around arterioles in the retina. This zone develops after vessels larger than primitive capillaries appear within the vascular complex, and it develops by a process of retraction or atrophy of immature capillaries or vascular mesenchyme surrounding developing arterioles. It makes its appearance one to several days after birth in the rat, and its significance is not entirely understood. While it is considered a feature of all species with retinal vessels, we have been unable to confirm its presence in the pig by either India ink studies or retinal digest preparations. The advantage conferred by having a few arterioles coming off of the major artery and feeding a capillary bed, rather than having numerous capillaries drain the arteriole is probably important in maintaining adequate pressure along the whole length of the arteriolar tree. It may be that the capillary-free zone is nothing more than a normal process of retraction of primitive capillaries to a distance from the arteriole equivalent to the diameter of the mesh of the capillary network, and this may be a general principle of vascular development. Indeed it has been shown in rabbit ear chamber experiments, that the loss of capillaries is greater in the neighborhood of developing arterioles than around developing venules.- 1 Our observations seem to place the development of the retinal vascular bed in context with the development of embryonic vessels elsewhere in the body. We feel that the study of retinal vasculogenesis may enhance the knowledge of vascular development in general. We would like to thank Miss Doris Pagan for secretarial assistance and Mr. Henrick Malpica for the illustrations. REFERENCES 1. Michaelson, I. C.: The mode of development of the retinal vessels and some observations of its significance in certain retinal diseases, Trans. Ophth. Soc. U. K. 68: 137, Michaelson, I. C.: Retinal circulation in man and animals, Springfield, 111., 1954, Charles C Thomas, Publisher. 3. Ashton, N.: Pathological basis of retrolental fibroplasia, Brit. J. Ophth. 38: 385, Ashton, N.: Oxygen and the growth and development of retinal vessels. In vivo and in vitro studies, XX Francis I. Proctor Lecture, Am. J. Ophth. 62: 412, Cogan, D. C: Development and senescence of the human retinal vasculature, Doyne Memorial Lecture, Trans. Ophth. Soc. U. K. 83: 465, Serpell, C: Polysaccharide granules in association with developing retinal vessels and with retrolental fibroplasia, Brit. J. Ophth. 38: 460, Nilhausen, K.: The vasoformative tissue in foetal retina with particular reference to the histochemical demonstration of its alkaline phosphatase activity, Acta ophth. 36: 65, Engerman, R. L., and Meyer, R. K.: Development of retinal vasculature in rats, Am. J. Ophth. 60: 628, Agrawal, P. K.: The cellular structure and development of the retinal vessels of the rat, Orient. A. Ophth. 3: 23, 1965.' 10. Engerman, R. L., Buesseler, J. A., and Meyer, R. K.: Periodic acid-schiff staining of retinal whole mounts, Arch. Ophth. 68: 62, Ashton, N.: Studies of the retinal capillaries in relation to diabetic and other retinopathies, Brit. J. Ophth. 47: 521, Ashton, N., and Blach, R.: Studies on developing retinal vessels. VIII. Effect of oxygen on the retinal vessels of the ratling, Brit. J. Ophth. 45: 321, McClure, C. F. W.: The endothelial problem, Anat. Rec. 22: 219, Arey, L. B.: The development of peripheral blood vessels, in Orbison, J. L., and Smith, D. E., editor: The peripheral blood vessels, Baltimore, 1963, Williams and Wilkins Company, p Shakib, M., and Oliveira, L. F.: Studies on developing retinal vessels. X. Formation of

11 530 Henkind and De Oliveira Investigative Ophthalmology October 1967 the basement membrane and differentiation of intramural pericytes, Brit. J. Ophth. 50: 124, Oliveira, L. F.: O isolamento dos capilares do cortex cerebral, Arq. port, oftal. 16: 87, Henkind, P.: Ballotini occlusion of retinal arteries. Collateral vessels, Brit. J. Ophth. 50: 482, Clark, E. R.: Studies on the growth of blood vessels in the tail of the frog larva, Am. J. Anat. 23: 37, Clark, E. R., and Clark, E. L.: Observations on living preformed blood vessels as seen in a transparent chamber in the rabbit's ear, Am. J. Anat. 49: 441, Ashton, N.: Retinal vascularization in health and disease, Am. J. Ophth. 44: 7 (II), Clark, E. R.: Growth and development of function in blood vessels and lymphatics, Ann. Int. Med. 9: 1043, 1936.