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In the few marsupial species studied to date that possess a retinal vasculature, the arterial and venous segments, down to the smallest calibre capillaries, have been shown to occur in pairs. It is a pattern seen in the marsupial central nervous system (CNS) but not in other tissues in this group or in any tissues in eutherian mammals. The aim of the present study was to investigate the presence of retinal vessels in a range of non‐eutherian mammalian species (marsupials and monotremes) and to determine if the pattern of paired vessels was a widespread phenomenon within this animal group.
Species studied included a monotreme, the short‐billed echidna (Tachyglossus aculeatus) and a range of Australian marsupials, the honey possum (Tarispedidae rostratus), fat‐tailed dunnart (Sminthopsis crassicaudata), grey‐bellied dunnart (Sminthopsis griseoventer), numbat (Myrmecobius fasciatus), broad‐footed marsupial mouse (Antechinus godmani) and the North American opossum (Didelphis virginiana). Eyes were fixed in glutaraldehyde or paraformaldehyde and retinas were embedded in resin for light and electron microscopic analysis.
Examination revealed that in those species with retinal vessels (fat‐tailed dunnart, grey‐bellied dunnart, numbat, marsupial mouse, North American opossum) the pattern of vessels differs from the conventional plexus‐like arrangement of mammalian retinal vasculature (that is, anastomotic networks of capillaries between arterioles and venules). In marsupials retinal vessels always occur in closely related pairs, with the arteriolar limb usually situated on the vitread aspect. Vessels penetrate the retina and branch to form layers of paired capillaries as far as the outer nuclear layer in some species. The capillaries form blind‐ended hairpin loops and display classical morphological features of CNS capillaries
The phylogenetic relations of this vascular pattern in the marsupial CNS and retina, and in the CNS of a few other classes of non‐mammalian vertebrates, suggest that retinal vascularisation may have evolved independently in marsupial and eutherian mammals and that the former may have evolved from a common primitive mammal‐like reptilian ancestor which possessed paired vasculature in the CNS. Eutherian mammals may have evolved from an ancestor with anastomotic networks in the CNS or this pattern may have evolved later in eutherian mammal evolutionary radiation. The possible functional and physiological significance of the paired vessels is discussed.
As blood vessels enter the parenchyma of the central nervous system (CNS) of marsupials they do so in distinctive pairs consisting of an artery and a corresponding vein which branch in unison down to, and including, the finest capillaries until the arterial and venous components of each limb unite forming a terminal arteriovenous hairpin capillary or end loop.1,2,3,4 The zones of CNS parenchyma supplied by such vascular units overlap little or not at all with neighbouring units and therefore all blood vessels in the marsupial CNS function as true end‐arteries.5 This differs from the conventional capillary anastomotic networks seen in most other tissues in marsupials and all tissues in eutherian mammals and monotremes.3,4,5
In some non‐mammalian vertebrates, the retina in addition to receiving nourishment from the choroidal vascular bed may also be supplemented by a range of extraretinal or intravitreal vascular structures such as the hyaloid system in amphibians, the pecten in birds or the conus in reptiles.6,7,8 In eutherian and non‐eutherian mammals, the temporary hyaloid vasculature which nourishes the developing lens and retina is considered the ontogenic remnant of the intravitreal vascular structures of their non‐mammalian ancestors. In adult eutherian mammals (all mammals except monotremes and marsupials) that have evolved more complex retinas and visual function the demand for additional supplementary retinal nourishment has resulted in the evolution of true intraretinal vascular networks or retinal vessels such as those seen in humans.6,7,8 In non‐eutherian mammals (monotremes and marsupials) both vascular (holangiotic), partially vascularised (paurangiotic) and avascular retinas are observed. The diprotodont group of marsupials (koala, wombats, possums, macropods (kangaroos, wallabies)) are generally described as possessing avascular retinas.8 The few marsupial species with a retinal vascular supply which have been studied3,9,10,11 belong to orders within the polyprotodont group. The intraretinal vessels in the few species studied to date have been described as being identical to the paired end artery vascular pattern found in the remainder of the marsupial CNS.2,3,9,10,11 Although these unusual paired retinal vessels were first described in the literature over 60 years ago, they have received little attention. Considering the efforts of so much research in the field of ophthalmology is aimed at understanding retinal vascular disease, it is surprising that such a radically different pattern of retinal vasculature has been so poorly described and is even less well understood. The aim of the present study was to provide an updated and more detailed description of this unusual form of retinal vascularisation in a range of previously undocumented or poorly documented species of non‐eutherian mammals. The evolution and functional significance of this pattern of vasculature is discussed.
Tissues from one monotreme (short‐billed echidna, Tachyglossus aculeatus) and a range of marsupials were investigated (see table 11).). All ocular tissues were collected from animals sacrificed as part of other investigations which conformed to institutional animal ethics committees and wildlife authority regulations. Tissues were fixed in glutaraldehyde or paraformaldehyde. Posterior segments or whole globes were processed and embedded in either Epon or Araldite resin in the standard fashion. Semithin sections (1–2 µm) were stained with toluidine blue. Ultrathin sections (50–70 nm) of selected regions were stained with uranyl acetate and lead citrate and examined using a Philips 410 transmission electron microscope. The methods of investigating the pattern of retinal vessels in Didelphis virginiana using vascular filling and wholemount preparations have previously been described.10 It was not possible to perform vascular filling experiments in the other species due to limited availability and access to live specimens.
Intraretinal vessels were present in both species of dunnart, the numbat, the marsupial mouse (or Atherton antechinus) and North American opossum (table 11).). The echidna and honey possum did not possess intraretinal vessels (fig 11).). Fundus or fluoroscein examinations were not performed on these latter two species therefore it was not possible to determine whether there were any vessels at the optic nerve head resembling a conus, a feature which has been described in some other marsupial species (table 22).9
Morphological features of the echidna eye, which were reptilian in character included an avascular rod‐dominated retina (fig 1A1A),), a keratinised corneal epithelium, an iris sphincter but no dilator or ciliary muscle, and cartilage plates within the sclera similar to that seen in reptiles and birds.21 A number of large calibre irregular‐shaped vessels with no supporting pericytes or intima or media were noted in the choroid of the echidna (not shown). Unlike other vessels whose lumina were clear as a consequence of the vascular perfusion, the lumina of the irregular vessels contained proteinaceous material. On the basis of the above features these vessels were identified as lymphatics which have so far not been described in the mammalian eye.
The distinctive features of the honey possum posterior segment included a heavily pigmented choroid and oil droplets in the cone‐rich retina (fig 1B1B).). The retinal thickness in the echidna and honey possum were approximately 110–125 μm.
The retinal vasculature in Didelphis virginiana (fig 2A2A)) has been described in detail previously.10 Briefly, 8–10 large radially arranged pairs of retinal vessels emerge from the optic disc. Each artery is closely paired with a companion vein which usually lies directly beneath or sclerad relative to the artery and is partially obscured in whole mounts viewed from the vitread aspect (fig 2B2B),), except near the optic disc where they may lie side by side for a short distance and also at the terminal capillary end loops where the paired nature of these vessels becomes evident (fig 2C2C).
Transverse semithin sections of the retina of adult North American opossum, numbat and both species of dunnart were characterised by the distinctive profiles of paired vessels of varying diameters ((figsfigs 2D, E, 3, 44)) and for the purpose of brevity will be described together except where species‐specific differences were noted. The larger retinal vessels in North American opossum (Didelphis virginiana) produce prominent elevations on the retinal surface, however in the numbat (Myrmecobius fasciatus) (fig 3A3A)) larger calibre vessels were more deeply located in the nerve fibre and ganglion cell layer (fig 3B, CC).). These larger retinal arteries demonstrated some uncharacteristic lamination of the elastic layer or basal lamina of the tunica media (fig 3B, CC),), a feature not usually present in normal retinal vessels in placental mammals. Vessels penetrating into the depths of the retina only rarely appear in transverse sections but are more apparent in oblique sections ((figsfigs 3D, 4C4C).). Paired retinal vessels branch repeatedly as they penetrate from the vitread to the sclerad aspect of the retina (fig 4A–C) and terminate in small capillaries which form hairpin end loops where the arterial and venous limbs become continuous ((figsfigs 2C, 4B–D). Thus without exception in the marsupial species investigated which were found to posses retinal vessels, these vessels form a complete system of end vessels that lack anastomoses even at their small capillaries (fig 2B, CC).
In whole mount preparations in Didelphis and in sections of all the species studied, several levels of capillaries were discernible in the retina ((figsfigs 3B, 4A, B, EE):): one layer in the nerve fibre layer or ganglion cells layer (fig 4A, C, EE)) and one in the inner plexiform or inner nuclear layer ((figsfigs 2, 3, 4B4B).). In Didelphis, a distinctive deeper layer is present within the outer nuclear layer extending almost as far as the external limiting membrane layer (fig 2E2E).). In most other species, at each of these levels, the capillaries run parallel to the retinal surface and appear most frequently as paired ovoid/circular profiles ((figsfigs 2D, 3E, 4A, B, EE).). In the grey‐bellied dunnart (Sminthopsis griseoventer) material examined paired vessels did not penetrate beyond the nerve fibre and ganglion cell layer (fig 4E4E).
In transverse sections of the species found to have paired vessels, the arterial and venous limbs of the closely apposed capillaries are indistinguishable, each being lined by a continuous thin endothelium and each surrounded by its own distinct basal lamina (fig 2E2E).). In oblique sections, the hairpin loops themselves were occasionally identified (fig 4E4E).). The retinal vascular endothelial cells are of normal non‐fenestrated retinal type with overlapping or mortise cell‐cell contact and tight junctions (fig 2E2E).). Numerous pericytes are associated with the paired capillaries but are unusual in that the nuclei are observed most frequently interposed between the two limbs of capillary loops and seldom in other regions of the capillary circumference ((figsfigs 2D, 2E, 3E, 4E4E).). The ocular tissue from broad‐footed marsupial mouse was poorly fixed and although allowed confirmation of the double vessel pattern did not permit more detailed morphological examination. A summary of the species investigated including their size, distribution, lifestyle, diet and habitat included in table 11.
Vessels of the choroid, as well as those found in other regions of the eye, in all the species examined do not form paired end‐loops, but rather form more conventional anastomosing capillary networks or large venous sinuses.
Since the early histological reports of the unusual form of paired vessels in the retina of marsupials3,18 little attention has been paid to the structure, function and evolutionary significance of this arrangement and whether it is a widespread phenomenon in marsupials. In light of the dearth of information on the vasculature of the marsupial eye, the present study aimed to survey the structure of the retina and supporting tissues in several species of marsupials and one monotreme (short‐billed echidna). The investigation revealed that the retinas of the insectivorous short‐billed echidna (Tachyglossus aculeatus) and nectiverous honey possum (Tarispedidae rostratus), both of which feed in daylight possess avascular retinas. Intraretinal vessels were present in the fat‐tailed dunnart (Sminthopsis crassicaudata), grey‐bellied dunnart (Sminthopsis griseoventer), numbat (Myrmecobius fasciatus) and broad‐footed marsupial mouse (Antechinus godmani) which all belong to orders in the polyprotodont group (that is, orders other than the diprotodontia; table 22).). Most of the species predominantly feed at dusk or dawn or are completely nocturnal with the exception of the numbat (table 11).). The thickness of the retina in those species possessing retinal vessels in the present study was over 143 μm, the value calculated to be the maximum thickness for avascular retinas which is a function of the diffusion characteristics of oxygen from the choroidal circulation to the retina.8,22 Those species with intraretinal vasculature demonstrated a pattern of vessels vastly different from the conventional plexus‐like arrangement of holangiotic retinas seen in eutherian mammalians, such as humans, where there are extensive networks of anastomotic capillaries between arterioles and venules. In marsupials the arteriolar limb and venous limb always occur in closely related pairs, with the former usually situated on the vitread aspect. Vessels penetrate the depths of the retina in this paired arrangement and branch to form layers of paired capillaries which finally terminate in hairpin end loops. The paired units rarely if ever overlap the area supplied by adjacent vascular pairs. These observations confirm and extend the initial observations of Wislocki3 and a limited number of other investigators.10,11
The similarity in the pattern of paired vessels to those in the brain and their shared structural characteristics is not surprising considering the embryological link between the eye and the brain.23 Experimentally induced emboli in the marsupial brain result in well‐circumscribed ischaemic zones which reflect the territory of individual capillary loops.5 Thus marsupial CNS capillaries are end arteries both anatomically and functionally, whereas in eutherian mammals although anastomoses exist in the capillary bed of the CNS they are still considered functionally (as are retinal vessels) to be end arteries. Various theories have been proposed to explain the possible functional and physiological basis for this unusual paired vascular pattern in the marsupial brain and retina. Possibilities include a countercurrent exchange system for thermoregulation or as a means of optimising retinal oxygen tensions.18,24 It has also been speculated that the paired arrangement may serve to conserve space occupied in the retina by vessels and thus minimise their interference with visual acuity, which seems unlikely because when viewed from the vitread aspect they create a similar profile to the vessels of eutherian holangiotic retinas.
More recently, on the basis of studies of retinal development in the fat‐tailed dunnart Rodger and co‐workers11 have proposed the paired vascular pattern may simply be related to the precocious development of marsupials which are required to interact—independent of maternal physiological support—with their environment much earlier than placental mammals. These authors demonstrated that retinal vessels in the dunnart develop as paired end artery loops that are patent “ab initio” and therefore can meet the immediate metabolic demands of the functional developing retina. In contrast to the events during retinal development in placental mammals the developing vasculature of marsupials does not have to undergo remodelling to become functionally patent.11 Thus a vasculature that functions as it grows would allow a young marsupial brain to regulate breathing, locomotion and navigate via olfaction to reach the pouch where it must suckle. This is a more attractive hypothesis than the previously discussed thermoregulation or space‐conserving theories.
The unusual paired vessels as seen in marsupial CNS tissue, including the retina, are present in the CNS of a few invertebrates including earthworms,25 lampreys but not other cyclostomes such as hagfish,12 almost no fish except possibly lungfish, a limited number of amphibians (newts, salamanders) and in a few reptiles including some lizards and sphenedon.12,26,27 Interestingly, some retiles appear to have a mixture of paired vessels and anastomotic networks or intermediate forms in their CNS.24 Paired vessels are not however present in the CNS of any birds, monotremes or eutherian mammals. This pattern of occurrence in the non‐vertebrates and non‐mammalian vertebrates suggests that one vascular pattern has evolved from the other several times in evolution.24
What does the presence of double vessels in the various marsupials tell us about their evolution? The mammalia include three major subclasses, the monotremes (Prototheria), marsupials (Marsupialia) and placental (Eutheria) mammals. Approximately 180 million years ago Pangea, the last supercontinent, began separating into two landmasses, Laurasia and Gondwanaland. That separation coincided with the last common ancestor of the mammalian clades (clade: a lineage of animals with a common genealogy). Continental drift would eventually isolate marsupials to Gondwanaland, especially Australia, Antarctica and South America. Once isolated on South America and Australia, around 30 million years ago by the submersion of the last dry land bridges linking Antarctica to Tasmania, monotremes and particularly marsupials continued to radiate and evolve into several different orders.28 Seven such orders are extant, three found in the new world: Didelphimorphia (common opossums); Microbiotheria (only one extant representative, the Monito del Monte (“little mountain monkey”, Dromiciops gliroides)), and Paucituberculata (shrew opossums). Four orders exist in Australia: the Dasyuromorphia (carnivorous marsupials such as dasyurids—for example, quolls, dunnarts, Tasmanian devils, and numbats), the Peramelemorphia (bandicoots and bilbies), the Notorycytemorphia (marsupial voles) and lastly Diprotodontia (koala, wombats, possums and macropods (kangaroos and wallabies)).28
Having inherited the double vessel pattern in the CNS from an early marsupial ancestor this pattern has been retained in all subsequent marsupial descendants. In marsupials that radiated into different niches and developed increasingly complex visual functions requiring a separate intraretinal vasculature, those vessels developed as paired vessels.10,11 Thus, meeting the functional needs of a more metabolically demanding inner retina with a true intraretinal plexus, rather than intravitreal vessels, such as a conus or pecten found in reptiles and birds, has led to the evolution of retinal blood vessels within marsupials in an almost identical manner to that which evolved independently in eutherian mammals. This illustrates that the imperative for such vascularisation was not a unique feature of eutherian mammal evolution. The evolution of intraretinal vessels is considered a more recent evolutionary adaptation associated with the radiation of mammal groups, including primates.8 To the author's knowledge, paired intraretinal vessels are present only in Dasyuromorphia and Didelphimorphia (table 22).). A large number of Australasian marsupials belonging to the order Diprotodontia, which includes kangaroos, wombats, the koala and most possums, have avascular retinas16,17,20 and are believed to have evolved from the didelphid‐dasyurid clade.29 This would suggest that retinal vessels may have been lost during the evolution of the diprotodonts as well as the bandicoots. The latter group, despite being classified with the polyprotodonts, lack true vascular retinas and instead exhibit a conus‐like vascular structure in the vitreous.19
In summary, regardless of whether marsupials independently evolved the paired end artery system in the CNS or have retained it from a primitive ancestral line, it is clear that marsupial groups that evolved higher visual functional requirements, and therefore required supplementary retinal nutrition, were destined to develop paired vessels typical of the remaining marsupial CNS.
In addition to those colleagues who supplied tissues (see table 1), the author would like to thank a number of undergraduate students who have undertaken small projects on comparative ocular anatomy over the last 10 years. These include Leonie Dyer, Grazyna Faux, Linh Nguyen, Vanessa Ryan, Nikhil Valvi, Haider Bangash, Nicola Bird, Rebecca Comerford, Danny Gould and Deborah Koh. The author is also grateful to Professor Dunlop, Dr Catherine Arrese and Dr Jenny Rodger for discussions on marsupial vision. The author wishes to thank Ivan Schwab for encouragement to continue to pursue my comparative ocular anatomy studies. Technical assistance was kindly provided by staff in the School of Anatomy and Human Biology including Mary Lee, Guy Ben Ary and Natascha Heuer.
CNS - central nervous system
Competing interests: None declared.