Light and Electron Microscopy Study of the Pecten Oculi of the Jungle Crow (Corvus Macrorhynchos)

Okajimas Folia Anat.Pecten Jpn., 87oculi(3): of 75–83, the jungle November, crow 201075

Light and electron microscopy study of the pecten oculi of the Jungle Crow (Corvus macrorhynchos)

Mohammad Lutfur RAHMAN1, 2, Eunok LEE1, Masato AOYAMA1 and Shoei SUGITA1

1 Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan 2 Department of Anatomy and Histology, Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University, Khulsi, Chittagong, Bangladesh

–Received for Publication, February 3, 2010–

Key Words: jungle crow, pecten oculi

Summary: In this study, the pecten oculi of a diurnally active bird, the Japanese jungle crow (Corvus macrorhynchos), was examined using light and electron microscopy. In this species, the pecten consisted of 24–25 highly vascularized pleats held together apically by a heavily pigmented ‘bridge’ and projected freely into the vitreous body in the ventral part of the eye cup. Ascending and descending blood vessels of varying caliber, together with a profuse network of capillaries, essentially constituted the vascular framework of the pecten. A distinct distribution of melanosomes was discernible on the pecten, the concentration being highest at its apical end, moderate at the crest of the pleats and lowest at the basal and lateral margins. Overlying and within the vascular network, a close association between blood vessels and melanocytes was evident. It is conjectured that such an association may have evolved to augment the structural reinforcement of this nutritive organ in order to keep it firmly erectile within the gel-like vitreous. Such erectility may be an essential prerequisite for its optimal functioning as well as in its overt use as a protective shield against the effects of ultraviolet light, which otherwise might lead to damage of the pectineal vessels.

Introduction type reported in the kiwi, the vaned type described in the ostrich, and the pleated type found in most birds (Meyer, The pecten oculi is a unique vascular and pigmented 1977; Martin, 1985). The pleated form of pectin oculi intraocular structure characteristic of the avian eye. Since has been most thoroughly investigated and is comprised the first correct description and interpretation made by primarily of an extensive network of blood vessels with Perrrault in 1676 (Sillman, 1973), this intraocular struc- intervening pigmented tissue. It arises from the optic disk ture has been the subject of investigation from various in the form of accordion pleats held together at the free aspects. Through experimental manipulation, analogy apical border by a heavily pigmented bridge of tissue and hypothesis, researchers have endeavored to assign (Seaman and Storm, 1963; Raviola and Raviola, 1967). plausible functions to this structure. These functions Considerable variation in the number of pleats and in the include: (1) intraocular pressure regulation (Seaman and size and shape of the pectin oculi exists in the pleated Himelfarb, 1963), (2) intraocular pH regulation (Brach, form of pecten within the avian species (Thomson, 1975), (3) stabilization of the vitreous body (Tucker, 1929). These variations are believed to be related to the 1975), (4) reduction of intraocular glare (Barlow and Os- behavior of birds in relation to their general activity and twald, 1972), (5) a blood and fluid barrier for the retina visual pattern (Braekevelt, 1988). Although the vascular and vitreous body (Rodriguez-Peralta, 1975), and, above framework of pecten has been studied by flat-mounting all, (6) a supplemental nutritive organ (Mann, 1924; following its severance from the underlying optic disk Meyer, 1977), a function which has been equivocally and apical bridge (Tanaka, 1938), routine histology (Bawa accepted by contemporary researchers. Grossly, pectin and Roy, 1974) and transmission electron microscopy oculi can be categorized into three types: the conical (TEM) (Seaman and Storm, 1963; Raviola and Raviola,

Corresponding author: Shoei Sugita, Laboratory of Function and Morphology, Department of Animal Science, Faculty of Agriculture, Utsunomiya University, 350, Mine-machi, Utsunomiya shi, Tochigi 321-8505, Japan. E-mail: [email protected] or [email protected] 76 M.L. Rahman et al.

1967; Braekevelt, 1988), the true framework of pecten, xylene, infiltrated in liquid paraffin and embedded in vis-a-vis the tissue components comprising the intraocu- wax. The embedded tissues were mounted on wooden lar structure in birds, has remained elusive. blocks and 7-µm-thick sections were cut using a mi- Light and transmission electron microscopic studies crotome. The sections were stained using hematoxylin have shown that the pecten oculi consists almost exclu- and eosin and viewed, and photographed under a light sively of blood vessels, extravascular pigmented cells and microscope. For Azan staining, the sections were depar- a superficial covering membrane (Meyer, 1977; Martin, affinized and shaken in azocarmine G solution (40°C) for 1985) that lacks both muscular and nervous tissue (Meyer, 30 minutes. The samples were treated in aniline alcohol 1977). The pecteneal blood vessels consist of arterioles, followed by acetic acid alcohol to adjust the color of the venules and highly specialized capillaries (Raviola and nuclei and the cell border. The sections were placed in Raviola, 1967) that freely anastomose with each other 5% 12 tungsto IV phosphoric acid n-hydrate solution for (Dieterich et al., 1973; Hossler and Olson, 1984; Matsu- 1 hour to 1.5 hours to assist the staining with aniline blue naga and Amemiya, 1990). The area between the vessels orange G. After dehydration and clearing, the coverslip is partly filled with pleomorphic pigment cells described was mounted using Canada balsam (MGK-S; Matsunami as melanocytes (Braekevelt, 1984) containing melanin Glass Ind., Kishiwada, Osaka, Japan). granules (Raviola and Raviola, 1967). As part of an ongoing comparative study of the supplementary retinal Immunohistochemistry for determination of the type of circulation and the pecten oculi in particular, this report collagen attempts to elucidate the organization of the pecten oculi The sections containing pecten were deparaffinized of the Japanese jungle crow (Corvus macrorhynchos), using xylene and then heated in an autoclave at 121 and compares and contrasts these findings with observa- °C for 20 minutes in citrate buffer (pH 7.4) to retrieve tions from other avian species. antigenicity (Shi et al., 1993). The sections were then cooled to room temperature and rinsed with phosphate- buffered saline (PBS; pH 7.4, 0.01 mol/L). To reduce Materials and Methods background staining, the sections were pre-incubated

with 3% hydrogen peroxide (H2O2) in methanol and Collection of the sample then in 10% normal goat serum in PBS, for 30 min each. Japanese jungle crows (Corvus macrorhynchos) of Rabbit polyclonal anti-chicken collagen 1 antibody (AbD either sex were collected by trapping in the city of Niiza, Serotec, Oxford, UK) was used at a concentration of 0.1 Saitama Prefecture. A total of nine normal jungle crows mg/mL. The specificity of this antibody for chick embryo with an average weight of 840 g were anesthetized by was confirmed in a previous study (Mauger et al., 1982). injection of pentobarbitone sodium (30 mg/kg) and per- The sections were incubated with anti-chicken collagen fused through the heart with warm (35°C) physiological 1 antibody at 4°C for 18 h. After they were rinsed in saline. This was followed by perfusion fixation with 1 PBS, all of the sections were incubated with a goat anti- L of Ringer solution (0.9% NaCl, 0.042% KCl, 0.025% rabbit antibody (ICN Pharmaceuticals, Costa Mesa, CA,

CaCl2 in distilled water, wt/vol) and 1 L of Zamboni fixa- USA) diluted 1:500. Immunoreactions were visualized tive (0.1 M phosphate buffer [pH 7.4] containing 2.0% by incubating the sections in Tris-HCL buffer contain- paraformaldehyde and 0.2% picric acid, wt/vol), before ing 0.02% 3’, 3′-diaminobenzidine (Dojin Laboratories, which the right auricle was cut open for drainage. After Kumamoto, Japan), 0.3% ammonium nickel (II) sulfate cessation of respiration and the heartbeat, the eyeballs hexahydrate and 0.015% hydrogen peroxidase for 10 min were removed from the orbital cavities. The cornea and and then dehydrating them in an ascending alcohol series lens were excised from the eyeball. The posterior half of and clearing them with xylene. the eyeball was then removed, and the vitreous body was washed carefully and immersed briefly in the Zamboni solution. The pecten oculi was carefully dissected out Transmission electron microscopic study and cut into smaller pieces for light and electron microscopy processing. The pecten was examined in both eyes, For transmission electron microscopy, the pecten and as fixation was judged to be good and no differences samples were sliced into 2 × 2 mm samples. The samples were noted between the two eyes, the sample was re- were fixed in 1% osmic acid solution for 1 hr, and then garded as valid. immersed in 2.5% glutaraldehyde for 1 hr. Then, the samples were dehydrated with ethanol and rinsed in 90% ethanol: 90% acetone (1:1 v/v), followed by 90%, 95% Light microscopy study and 100% acetone. The samples were then immersed in Epon 812 with absolute acetone (1:1 v/v, overnight), fol- Hematoxylin and Eosin (H&E) and Azan staining lowed by Epon 812 (overnight). The immersion in Epon Intact fixed pectens were dehydrated, cleared using 812 was repeated twice under vacuum in a desiccator for Pecten oculi of the jungle crow 77

3–4 hrs. The samples were placed into molds, and the side of the trapezoid (Fig. 1). The pecten looked like a blocks were cured in an oven at 60°C for 24 hr. Ultrathin thin, pleated lamina in a transverse section. The pleats of sections (70-nm thick) were cut using an Ultracut S ul- the pecten appeared in the form of an accordion pattern. tramicrotome (Reichert-Jung Optische Werke AG, Wien, Abundant blood capillaries and a few afferent and effer- Austria) equipped with a diamond knife (Diatome AG, ent blood vessels were observed (Fig. 2). The capillaries Biel, Switzerland). Sections were placed on a 200-mesh were generally arranged in two layers and surrounded the copper grid, stained with 2% uranyl acetate in 50% etha- larger vessels (Fig. 2). The larger vessels and capillaries nol, and post-stained with 0.2% lead citrate. The sections were provided with a thick endothelium and a prominent were observed using a transmission electron microscope adventitial coat. The lumen of the vessels was occupied (H-800; HITACHI, Tokyo, Japan) under an accelerating by numerous erythrocytes (Figs. 3, 4). Cells containing voltage of 100 kV at a magnification of ×3,000–6,000. granules of melanin occupied the grooves between the To calibrate the images, the resulting micrographs were vessels, and thus smoothed out the surface of the lamina scanned at 300 d.p.i. using an Epson GT X-700 flatbed (Figs. 2, 3, 4). The bridge was crescent-shaped in vertical scanner. sections of the pecten. Its surface was often provided with narrow depressions, occasionally deep and tortuous, that afforded attachment for fibers of the vitreous body Results (Fig. 2). The bridge consisted mainly of a network of anastomosing cords of pigment cells. The meshes of the The pecten of the crow was a trapezoid fan-like network were occupied by capillaries, some of which had intraocular structure and was black or brown in color a thick or thin endothelial lining (Fig. 4). (Fig. 1). The base of the trapezoid inserted upon the head Serial sections through the base of the pecten and the and the cauda of the optic nerve, and the rest projected underlying cauda of the optic nerve demonstrated that into the vitreous body. It consisted of a pleated lamina, these vessels arose from the basal artery of the pecten 120 to 348 µm in thickness, whose sinuous course was (Fig. 5). They must therefore be regarded as afferent fixed distally by its attachment to a compact cord of vessels. The second type of vessel ranged from 5 to 80 pigmented tissue, “the bridge,” running like a handrail µm in diameter and was provided with an endothelium along the free margin of the organ. The pectineal pleats 2 to 3 µm thick (Fig. 5). Capillaries 20 µm or less in were separated by alternating depressions, and the base diameter were preponderant, while large vessels with a was wider than the apical end (Fig. 1). The number of thick endothelium numbered only 2 or 3 in each fold and pleats varied from 24 to 25, one or two of which did not were usually located at the edges. Serial sections through reach the distal edge and merge with the dorso-temporal the base of the pecten and the underlying ocular tunics

Fig. 1. Lateral view of the pecten oculi of the jungle crow (Corvus macrorhynchos) illustrating the pectineal pleats (P) separated by alternating depressions (arrowhead). The base (B) is wider Fig. 2. Transverse section of the pecten appearing as a thin, pleated than the apical end. The pleats arise from the base and attach to lamina. The pleats appear in the form of an accordion pattern. the bridge (Br). Note the thickenings at the apical ends of each Numerous blood capillaries (C) and a few afferent (arrow) and pectineal pleat, the blood vessels appearing in the form of mid- efferent (arrowhead) blood vessels are observed. The capillar- rib-like prominences, and the ill-defined prominences on the ies are generally arranged in two layers and surround the larger surface (arrows). Bar: 5 mm. vessels. H&E stain. Bar: 100 µm. 78 M.L. Rahman et al. showed that the largest vessels of the pecten, which were efferent. Under a light microscope, the walls of the had a thick endothelium in the lamina, acquired a thin capillaries and the efferent vessels appeared similar. The endothelial lining at the base of the pecten, then passed vessels occasionally anastomosed to form the ampulla through the bundles of the optic nerve fibers, and finally (Fig. 7). The ampulla was more frequently located to- joined a large vein situated in the choroid, immediately wards the apical part of the pleats. Within the ampulla, beneath and parallel to the cauda of the optic nerve (Fig. the intercapillary spaces were occupied by pigment cells. 6). Therefore, the large vessels with thick endothelium The diameter of an ampulla was 100 to 200 µm (Fig. 7).

Fig. 3. Light micrograph of a large blood vessel in the Fig. 4. Light micrograph illustrating a portion of the heavily pigmented bridge pecten oculi of the crow (Corvus macrorhynchos), (Br) and the vascularized pleated lamina. Note the tightly packed network illustrating the attenuated endothelium (arrow) and of pigment cells (P) and the limited number of capillaries (C) within the the thick basement membrane (M). Erythrocytes bridges. H&E stain. Bar: 50 µm. (arrowheads). H&E stain. Bar: 50 µm.

Fig. 5. Oblique section through the insertion of the pecten upon the Fig. 6. Serial section of the same specimen shown in Fig. 5. A venule optic nerve cauda (ONC). A basal artery (Ar) is lodged in a (Ve) is present at the edge of a fold in the pecten. The vein (V) groove on the vitreal surface of the mass of optic nerve fibers. has now reached the base of the pecten and merged toward the An artery (A) is present with the regular distance of a fold in the deep choroidal vein. ONC: Optic nerve cauda; Re: retina. Azan pecten. Re: retina. Azan stain. Bar: 100 µm. stain. Bar: 100 µm. Pecten oculi of the jungle crow 79

The wall of the vessels in the pecten was composed of was delineated by a fine basal lamina (Figs. 9, 10). The unspecialized endothelial cells, a single sheath of smooth basal lamina of the pecten capillaries was unusually muscle cells and a thick adventitia. The inner region of thick, averaging 0.5 µm with areas of up to 1.5 µm in the adventitia mainly consisted of layers of basement thickness (Figs. 9, 10, 11). The basal lamina consisted of lamina-like materials, while the outer region was rich in several fibrillar layers, each of which had the appearance collagen fibrils type I (Fig. 8). of a thin basal lamina. The melanocytes of the pecten Electron microscopy revealed that the entire pecten were large pleomorphic cells with long projections that

Fig. 7. Light micrograph illustrating a capillary ampulla (Am) (arrow). Fig. 8. Immunohistochemical staining revealing collagen type I Note the tightly packed network of pigment cells within the (arrow) fibers in the basement lamina of the capillaries. Bar: capillaries. Azan stain. Bar: 100 µm. 100 µm.

Fig. 9. Electron micrograph indicating the multilayered basal lamina Fig. 10. Electron micrograph along the capillary and its peripheral (Bl) of the capillaries, the lumen of the capillaries (Lu), and areas, showing the nuclear regions (N) of melanocytes (M). The nuclear regions (N) of melanocytes. ×3200. multilayered basal lamina (Bl) of the capillaries is indicated. Lu: lumen of the capillaries. ×3200. 80 M.L. Rahman et al.

Fig. 11. Electron micrograph showing numerous fine microfolds that Fig. 12. Electron micrograph showing numerous fine microfolds on branch and anastomose (arrowhead). A typical endothelial cell both the luminal (L) and abluminal (Ab) borders of the endo- with a thin cell body (arrow) and thicker nuclear region (N). thelial cells of a capillary (C). The covering membrane (Cm), “Ab” or “L” indicates the abluminal or luminal borders of the nucleus (N) of endothelial cells and pericyte (P) are also indi- endothelial cells of a capillary (C), respectively. Bl: The basal cated. ×4000. lamina of the capillaries. ×4000.

more or less isolated the capillaries from one another. Discussion The nucleus of the melanocytes was large and vesicular (Figs. 9, 10). Melanocytes were most abundantly located In birds where the retina is avascular, the pecten, peripherally in the pecten immediately below the basal a highly vascularized intraocular organ with sparse lamina (Figs. 9, 10), but they could also be observed pigmented tissue, is strongly suspected of playing a more centrally within the pecten. The capillaries of the nutritive role (Duke-Elder, 1958; Meyer, 1977). A tropic crow pecten were extremely infolded on both the luminal role calls for an expansive surface area of the diffusion and abluminal borders (Figs. 11, 12). In many cases, surfaces and a high blood supply. This must, however, the actual cell body was only a thin central area from be achieved after meeting certain constraints: the pecten which numerous projections arose (Figs. 11, 12). These must not be too large to interfere with the optical func- processes were thought to be microfolds, rather than tion of the eye and must also be stable enough to resist microvilli, as they exhibited a range of widths when cut lateral swings. Our present observation of the pecten of in different planes (Figs. 11, 12). The microfolds project- the jungle crow, Corvus macrorhynchos, and findings ing into the lumen were longer (1.0 to 2.0 µm) than those from a study of pectens from birds with diverse habitats formed on the abluminal surface, which ranged from 0.6 and differing visual acuity (Kiama et al., 2001) suggest to 0.8 µm in length (Figs. 11, 12). It is difficult, however, that the pecten has been designed to overcome the above to obtain true measurements of the lengths of these folds, constraints. The jungle crow is a largely diurnal bird as they are often extremely tortuous in shape. On both of prey, exhibiting a cosmopolitan distribution. Shlaer the luminal and abluminal borders, these microfolds (1976) reported that birds of prey have keen vision, and often appeared to branch and anastomose (Fig. 11). In our previous observations regarding the visual capacity the capillary endothelial cells, the nuclear region was the of the jungle crow (Rahman et al., 2006) also support widest portion of the cell body (Fig. 11). Pericytes were this notion. The pecten oculi has been considered to often enclosed within the thick compound basal lamina play a major role in the acquisition of an efficient visual of the capillaries (Fig. 12). The pericytes did not display mechanism (Mann, 1924). The location of the pecten in microfolds (Fig. 12). The pericytes might be separated the jungle crow conforms to that evident in other diurnal from the endothelial cells by basal lamina materials species of birds possessing a pleated type of pecten. It (Fig. 12), or they might be in apparent contact with the lies over the optic disc, and the free end projects into the abluminal folds of the endothelial cells, with no interven- vitreous, as shown in the chick (Bhattacharjee, 1993). ing basal lamina layers (Fig. 12). The wall of the blood Since the optic disc constitutes a blind spot on the retina, vessels consisted of a thin nonfenestrated endothelium it is of great advantage for the pecten to also be located surrounded by a basal lamina of much the same thickness in a position that optimizes the retinal area available and appearance as that described for the pecten capillar- for the capture of images. Although the location of the ies (Figs. 11, 12). pecten is the same in all birds so far studied, the specific characteristics of the organ, such as its size, form and the Pecten oculi of the jungle crow 81 number of pleats, vary greatly (Thomson, 1929; Meyer, microfolds is very small (normally between 1.0 and 1.5 1977). Other than the pleated type, there are two other µm), this may indicate the optimum size of microfolds morphological types of pecten (Meyer, 1977), e.g., the or may perhaps represent the tallest microfolds that can conical type and the vaned type. The conical pecten is a be supported adequately by the endothelial cells. Another finger-like structure resembling the conus papillaries of striking feature of pecten capillaries is the unusually reptiles and is found only in the kiwi bird (Apteryx man- thick basal lamina described in all species to date (Meyer, telli) (Meyer, 1977). The vaned pecten consists of a cen- 1977, Braekevelt, 1984, 1986, 1988, 1990, 1991). This tral flattened pillar from which vertical vanes arise. This thickened basal lamina might appear incongruous associ- type of pecten is found in the ostrich and the rheas (Walls, ated with capillaries so obviously involved in transport 1942; Meyer, 1977). The pleated pecten is by far the functions. However, despite its overall thickness, the most common type of pecten, with variations found in all fibrillar layers of the basal lamina are not closely packed, other birds. These variations are believed to depend on and the entire structure would not appear to offer a seri- the behavior of birds in relation to their general activity ous barrier to the movement of materials. It is speculated and visual pattern (Kiama et al., 2001). A basal lamina or that this thickened basal lamina may actually serve an vitreo-pecteneal limiting membrane continuous with the important structural function, as it supports fragile endo- inner limiting membranes of the retina covers the entire thelial cells that have very thin cell bodies and numerous pecten (Braekevelt, 1986, 1988, 1990, 1991). In some processes. The pecten of the Japanese jungle crow was species, hyalocytes are a regular feature adherent to the relatively large and the basal laminae were relatively outer surface of this membrane (Braekevelt, 1984, 1990). thick. Similarly thick basal laminae have been reported Within the folds of the pecten were located many special- to be present in the larger pectens of the great blue heron, ized capillaries, supply (afferent) and drainage (efferent) red tailed hawk, loon and pigeon (between 1.0 and 2.0 vessels and numerous large, branching melanocytes. µm), compared with the thinner basal lamina in the Unlike the chicken (Dieterich et al., 1973) and pigeon smaller pecten of the nighthawk (0.5 µm) and the basal (Raviola and Raviola, 1967), where the authors could lamina with intermediate thickness in the intermediate- differentiate arterioles and venules in the pecten, in the sized pecten of the mallard (0.75 µm), possibly reflecting crow, as in all previous studies of other species by these this structural role (Braekevelt, 1984, 1988, 1990, 1991). authors, it is very difficult if not impossible to adequately Pericytes, which are a common feature of the wall of categorize these larger vessels in the pecten as to being both retinal and hyaloid capillaries, are also present either arterioles or venules (Braekevelt, 1984, 1986, in the walls of pectineal capillaries (Ashton and De 1988, 1990, 1991). This apparent lack of structural dif- Oliviera, 1966; Braekevelt and Hollenberg, 1970; Jack, ference between most of these supply and drainage ves- 1972). Similar to the function of these cells in other loca- sels within the body of the pecten may indicate a lowered tions, the function of the pericytes in pecten capillaries blood pressure within the pecten. The capillaries within is uncertain, and they may be supportive or contractile in the avian pecten are extremely specialized vessels; one nature or may perhaps represent reserve cells that could of the striking features of these capillaries is the presence become endothelial cells, as required. of a huge array of long processes on both their luminal The present study demonstrated how a large con- (apical or internal) and abluminal (basal or external) glomerate of pectineal blood vessels of varying caliber borders. While the authors of some ultrastructural stud- is effectively arranged and reinforced by an extensive ies have referred to these as microvilli (Nguyen et al., contingent of melanosomes. Although investigators have 1967), most other investigators have referred to them as attributed a supportive role to the pigmented intervas- microfolds (Dieterich et al., 1973; Meyer, 1977). In the cular tissue of the pecten in TEM studies (Braekevelt, present study in Japanese jungle crows, these processes 1991), the functional relationship between the melano- were thought to be microfolds, rather than the finger- cytes and the pectineal vessels has remained unresolved. like structure implied by the term microvilli (Braekevelt, Scrutiny of the figures presented in this study raises the 1984, 1986, 1988, 1990, 1991). The microfolds on the possibility of a two-fold causal relationship between luminal surface always appeared to be longer, straighter the two components of pectineal tissue: (1) structural and more numerous than those on the abluminal edge, reinforcement of the vascular lattice, and (2) shielding of perhaps indicating enhanced transport out of the capil- the blood vessels from exposure to ultraviolet light. The laries. While some variation in the height of both the structural reinforcement of the pectineal blood vessels luminal and abluminal folds has been reported across is important for maintaining the erectile function of the species, these variations do not seem to be correlated pecten and protecting the visual efficiency of the eye. with the overall size of the pecten, as even the relatively This aspect of melanocytic function is provided by the small pecten of the night hawk shows microfolds similar perikarya of the melanocytes as well as by the melanin in height to those reported in species with larger pectens, granules contained within. Whereas the perikarya of the such as those found in the great blue heron (Braekevelt, melanocytes lie wedged between adjoining blood vessels, 1984, 1991). Since the range of heights of the luminal the melanosomes, which occur in sizeable numbers, plug 82 M.L. Rahman et al. into the interstices of the vascular network in different half of the pecten and the lateral aspect of the pectineal conformational patterns to reinforce the pecten at differ- pleats, where melanocytes are much fewer and the capil- ent locations. This may account for the differential distri- lary network of blood vessels is partially exposed. bution of melanosomes in the pecten. On the free apical half of the pectineal surface, the melanosomes formed a thick investment over the anastomosing network of Acknowledgments blood vessels, giving the latter a bloated appearance. The melanosomes at this part of the pecten appeared to pro- This work was supported by a Grant-in-Aid for vide protection for the ensheathed blood vessels against Scientific Research from the Ministry of Education, Sci- exposure to incident ultraviolet light. The great prepon- ence, Culture, Sports and Technology of Japan (project # derance of melanosomes at the apical end of the pecten 19208029) and from the Japan Society for the Promotion tends to implicate a similar melanosomal function. Such of Science (JSPS) (# 20-08111). an inference seems plausible in view of the increase in the number of melanosomes in the keratinocytes of man following exposure to ultraviolet light (Ghadially, 1982). References Furthermore, melanin is an efficient absorber of light: this is a non-specific phenomenon and extends through 1) Ashton N and Oliveira. Nomenclature of pericytes-intramural and extramural. Br J Ophthal 1966; 50:119–123. the ultraviolet region into the visible range, but it is 2) Barlow HB and Ostwald TJ. Pecten of the pigeon’s eye as an most pronounced toward the shorter end of the spectrum inter-ocular eye shade. Nat New Biol 1972; 236:88–90. (Jimbow et al., 1991; Kollias et al., 1991). 3) Bawa SR and Yashroy RC. Structure and function of vulture When tissues are exposed to reactive oxygen radicals, pecten. Acta Anat (Basel) 1974; 89:473–480. a variety of pathological changes may occur such as 4) Bhattacharjee J. A presumably astroglial cell in the retino- pectineal junction in Gallus domesticus, demonstrated by non- protein denaturation, lipid peroxidation and cell degen- specific esterase staining. Eur J Morphol 1993;31: 169–173. eration (Fridovich, 1978). The eye is no exception, with 5) Brach V. The effect of intraocular ablation of the pecten oculi of reported conditions including cataract (Van-Kuuk, 1991; the chicken. Invest Ophthalmol 1975; 14:166–168. Xiao-Lan and Marjorie, 1993) and retinal degeneration. 6) Braekevelt CR. Electron microscopic observations on the pecten The vertebrate retina is believed to be particularly sus- of the great blue heron (Ardea herodias). Histol Histopatho 1991; l6:345–351. ceptible to this kind of damage because (1) it has a high 7) Braekevelt CR. Fine structure of the pecten oculi of the mallard oxygen tension (Weiter, 1987), and (2) it is exposed to (Anas platyrhynchos). Can J Zool 1990; 68:427–432. ultraviolet light, which has been shown to initiate oxygen 8) Braekevelt CR. Fine structure of the pecten of the pigeon radical formation (Delmelle, 1979). This lends further (Columba livia). Ophthalmologica 1988; 196:151–159. support to the envisaged function of melanosomes as 9) Braekevelt CR. Fine structure of the pecten oculi of the common loon (Gavia immer). Can J Zool 1986; 64:2181–2186. a protective shield against sustained daytime exposure 10) Braekevelt CR. Electron microscopic observations on the pecten to ultraviolet light. Moreover, in view of the existing of the nighthawk (Chordeiles minor). Ophthalmologica 1984; notion that melanin reacts with excited molecules and 189:211–220. radicals (Foote, 1982; Larsson, 1993), a close association 11) Braekevelt CR and Hollenberg MJ. Comparative electron micro- between melanin-bearing melanocytes and the pectineal scopic study of development of hyaloid and retinal capillaries in albino rats. Amer J Ophthal 1970; 69:1032–1046. blood vessels in the jungle crow may be looked upon as a 12) Delmelle M. Possible implication of photo-oxidation reactions in strategy to protect the integrity of the capillary endothe- retinal photo-damage. J Photochem Photobiol 1979; 29:713–716. lium. 13) Dieterich CE, Dieterich HJ, Spycher MA and Pfautsch M. Fine A high degree of pigmentation of the pectineal bridge structural observations of the pecten oculi capillaries of the has also been reported in chicks (Seaman and Storm, chicken. Freeze-etching, scanning and transmission electron micro scopic investigations. Z Zellforsch Mikrosk Anat 1973; 146: 1963; Bhattacharjee, 1993), pigeons (Raviola and Ravi- 473–489. ola, 1967) and vultures (Bawa and Roy, 1974). Bawa and 14) Duke-Elder and Sir S. System of ophthalmology, The eye in evo- Roy suggested that this would favor light absorption and lution, Vol. I. Kimpton, London, 1958. a consequent increase in pectineal temperature, leading 15) Foote CS. Light, oxygen and toxicity. In: P. Author, ed, Pathology to an increase in the physiological activity of this intra- of oxygen Academic Press, New York, 1982; 21–44. 16) Fridovich I. The biology of oxygen radicals. Science 1978; 201: ocular structure in the transport of nutrients to the retina. 875–880. However, it should be pointed out that the close associa- 17) Ghadially FN. Melanosomes. Ultrastructural pathology of the cell tion between the melanosomes and the capillaries, and and Matrix, 2nd edition, Butterworths, London: 1982, pp.599–623. the differential distribution of the melanosomes on the 18) Hossler FE and Olson KR. Microvasculature of the avian eye: pectineal surface, as demonstrated in this study, cannot studies on the eye of the duckling with microcorrosion casting, scanning electron microscopy, and stereology. Am J Anat 1984; be completely explained by the provision of a rise in tem- 170:205–221. perature in the pecten alone, although such a possibility 19) Jack RL. Ultrastructure of the hyaloid vascular system. Arch cannot be excluded. The nutritive role of the pecten may Ophthal 1972; 87:555–567. possibly be mediated more efficiently along the basal 20) Jimbow K, Fitzpatrick TB and Wick MM. Biochemistry and Pecten oculi of the jungle crow 83

physiology of melanin pigmentation. In: Goldsmith LA, ed, The 32) Rodriguez-Peralta LA. Hematic and fluid barriers of the retina physiology, biochemistry, and molecular biology of the skin, 2nd and vitreous body. J Comp Neurol 1975; 132:109–124. ed Oxford University Press, New York, 1991, pp.873–909. 33) Seaman AR and Himelfarb TM. Correlated ultrafine structural 21) Kiama SG, Maina JN, Bhattacharjee J and Weyrauch KD. changes of the avian pecten oculi and ciliary body of Gallus Functional morphology of the pecten oculi in the nocturnal spot- domesticus. Am J Ophthalmol 1963; 56:278–296. ted eagle owl (Bubo bubo africanus), and the diurnal black kite 34) Seaman AR and Storm H. A correlated light and electron micro- (Milvus migrans) and domestic fowl (Gallus gallus var. domesti- scope study on the pecten oculi of the Domestic fowl (Gallus cus): a comparative study. J Zool Lond 2001; 354:521–528. domesticus). Exp Eye Res 1963; 2:63–172. 22) Kollias N, Sayre RM, Zeise L and Chedekel MR. Photo protection 35) Shi S, Chaiwun B, Young L, Cote JR and Taylor RC. Antigen by melanin. J Photochem Photobiol B 1991; 9:135–160. retrieval technique utilizing citrate buffer or urea solution for 23) Larsson BS. Interaction between chemicals and melanin. Pigment immunohistochemical demonstration of androgen receptor in Cell Res 1993; 6:127–133. formalin-fixed paraffin sections. J Histochem Cytochem 1993; 24) Mann IC. The function of the pecten. Br J Ophthalmol 1924; 8: 41:1599–2004. 209–226. 36) Shlaer R. An eagle’s eye: Quality of the retinal image. Science 25) Martin RG. In: Kings AS and McLeland J, eds, Form and function 1976; 176:920–922. in birds, Vol. 3. Academic Press Inc, London, 1985, pp.311–373. 37) Sillman AJ. Avian vision. In: Farner S, King JR and Parkes KC, 26) Matsunaga N and Amemiya T. Pecten of the chick eye demon- eds, Avian Bioligy, Vol.111 Academic Press, New York, 1973, strated by vascular casts. Okajimas Folia Anat Jpn 1990; 67:263– pp.349–387. 270. 38) Tanaka H. On the pecten of Japanese birds. Jap J Med Sci 1938; 27) Mauger A, Demarchez M, Herbage D, Grimaud JA, Druguet M, 7:95–132. Hartmann D and Sengel P. Immunofluorescent localization of 39) Thomson A. The pecten, considered from an environmental point collagen types I and III, and of fibronectin during feather morpho- of view. Ibis 1929; 12:608–639. genesis in the chick embryo. Dev Biol 1982; 94:93–105. 40) Tucker R. The surface of the pecten oculi in the pigeon. Cell 28) Meyer DB. The avian eye and its adaptations. In: Crescittelli F, Tissue Res 1975; 157:457–465. ed, In handbook of sensory physiology, VII/5, The visual system 41) Van-Kuuk FJGM. Effect of ultraviolet light on the eye: role of in vertebrates, Springer, Berlin, 1977, pp.549–512. protective glasses. Environ Health Perspect 1991; 96:177–184. 29) Nguyen J, Delaville A and Coujard R. L’ endothelium capillaire 42) Walls GL. The vertebrate eye and its adaptive radiation. Cran- du peigne de I’ oeil des oiseaux. Z Micr Anat Forsch 1967; brook Press. Bloomfield Hills. 1942. 77:432–441. 43) Weiter JJ. Phototoxic changes in the retina. In: Miller D, ed, 30) Rahman ML, Sugita S, Aoyama M and Sugita S. Number, dis- Clinical light damage to the eye, Springer, New York, 1987, pp.77 tribution and size of ganglion cells in the retina of jungle crow –125. (Corvus macrorhynchos). Anat Sci Int 2006; 81:253–259. 44) Xiao-Lan C and Marjorie FL. The effect of recovery of long term

31) Raviola E and Raviola G. A light and electron microscopic study H2O2 exposure on lens morphology and biochemistry. Exp Eye of the pecten of the pigeon eye. Am J Anat 1967; 120:427–461. Res 1993; 57:157–167.