Morphological and Morphometric Study of the Pecten Oculi in the Budgerigar (Melopsittacus undulatus).код для вставкиСкачать
THE ANATOMICAL RECORD 295:540–550 (2012) Morphological and Morphometric Study of the Pecten Oculi in the Budgerigar (Melopsittacus undulatus) ANTONIO MICALI,1 ANTONINA PISANI,1 CLAUDIA VENTRICI,1 DOMENICO PUZZOLO,1* ANNA MARIA ROSZKOWSKA,2 ROSARIA SPINELLA,2 AND PASQUALE ARAGONA2 1 Department of Biomorphology and Biotechnologies, Section of Histology and Embryology, University of Messina, Policlinico Universitario, Via Consolare Valeria 1, I-98125, Messina, Italy 2 Department of Surgical Specialties, Section of Ophthalmology, University of Messina, Policlinico Universitario, Via Consolare Valeria 1, I-98125, Messina, Italy ABSTRACT The pecten oculi is a highly vascular and pigmented organ placed in the vitreous body of the avian eye. As no data are currently available on the morphological organization of the pecten in the Psittaciformes, the pecten oculi of the budgerigar (Melopsittacus undulatus) was studied. The eyes from adult male budgerigars were examined by light, transmission, and scanning electron microscopy and a morphometric study on both light and transmission electron microscopy specimens was also performed in the different parts of the organ. In the budgerigar, the type of the pecten oculi was pleated. Its basal part had a cranio-caudal and postero-anterior course; its body consisted of 10–12-folds joined apically by a densely pigmented bridge. The pecten showed many capillaries, whose wall was thick and formed by pericytes and endothelial cells. These latter had a large number of microfolds, rectilinear on their luminal surface and tortuous on their abluminal surface. Interstitial pigment cells were placed among the capillaries, ﬁlled with melanin granules and showed many cytoplasmic processes. The morphometric analysis demonstrated signiﬁcant differences among the three parts of the organ relative to the length of the endothelial processes and to the number and size of the pigment granules. The morphological and morphometric analysis showed that the bridge of the budgerigar, different from the other birds, had a large number of capillaries, so that this part of the organ could also play a trophic role for the retina in addition to the choriocapillaris. C 2012 Wiley Periodicals, Inc. Anat Rec, 295:540–550, 2012. V Key words: pecten oculi; capillaries; endothelial cells; pigment cells; morphometry; Melopsittacus undulatus INTRODUCTION The pecten oculi is a highly vascular and heavily pigmented structure, placed in the vitreous chamber, along the course of the fetal ﬁssure of the eye of all examined avians. It is considered as an indirect retinal trophic system (Michaelson, 1954), as it supplements via the vitreous humor the choriocapillaris, the only constant retinal direct system observed in vertebrates (Puzzolo, 1994). The pecten can show three different morphological patterns. The conical type, observed only in the paleognath kiwi (Rochon-Duvigneaud, 1943; Meyer, 1977), C 2012 WILEY PERIODICALS, INC. V *Correspondence to: Domenico Puzzolo, M.D., Department of Biomorphology and Biotechnologies, University of Messina, Torre Biologica—Policlinico Universitario, Via Consolare Valeria 1, I-98125, Messina, Italy. Fax: þ39 90 692449. E-mail: email@example.com Received 24 September 2011; Accepted 4 January 2012 DOI 10.1002/ar.22421 Published online 20 January 2012 in Wiley Online Library (wileyonlinelibrary.com). THE PECTEN OCULI OF THE PARROT resembles the papillary cone of the lacertilia (Micali et al., 1988) and shows a cylindrical shape. The vaned type, typical of other paleognaths, such as the ostrich (Kiama et al., 2006) and the rheas (Meyer, 1977), is formed by a central pillar, around which many vertical and thin lamellae are placed. The pleated type, found in a large number of neognaths, is well studied either during its development (Puzzolo et al., 1980; Parducci et al., 1987; Uehara et al., 1990; Liebner et al., 1997) or in the adult (Fischlschweiger and O’Rahilly, 1966; Raviola and Raviola, 1967; Fielding, 1972; Jasinski, 1973; Dieterich et al., 1973; Bawa and YashRoy, 1974; Dieterich and Dieterich, 1974; Braekevelt, 1984, 1988, 1994, 1998; Puzzolo et al., 1985a; Kiama et al., 1994, 1997, 1998; Braekevelt and Richardson, 1996; Smith et al., 1996; Scala et al., 2002; Rahman et al., 2010; Gültiken et al., 2011). The pleated type is composed of three different parts: the base, adjacent to the neuroretina, the folds, arranged like an inverted fan, and the bridge, running like a handrail along the vitreous margin of the organ. Structural and ultrastructural studies have demonstrated the constant presence of a superﬁcial limiting membrane covering the entire pecten (Braekevelt, 1998), of hyalocytes (Uehara et al., 1996), of a large number of interstitial pigment cells (Dieterich and Dieterich, 1974), and of many capillaries; these latter are surrounded by a thick basement membrane (Braekevelt, 1984; Corona et al., 2004) and formed by endothelial cells with long microfolds on both luminal and abluminal surfaces (Braekevelt, 1988; Scala et al., 2002) and by some abluminal pericytes (Raviola and Raviola, 1967; Rahman et al., 2010). The size and the shape of the pecten and the number of its folds showed peculiar variations (Braekevelt, 1988), which were considered independent to the eye size but were rather related to the environmental lighting (Bawa and YashRoy, 1972) and to the activity of the bird (Kiama et al., 2006). No data are currently available on the ﬁne morphology of the pecten in the order of Psittaciformes, such as the budgerigar Melopsittacus undulatus (Shaw, 1805), a largely available and easily handling species. In this work, we provide morphological and morphometric data on the pecten oculi of this bird to demonstrate its structural and ultrastructural organization and to present the ﬁrst morphometric data on the blood vessels and on the pigment cells in the different parts of the organ. MATERIALS AND METHODS The work was carried out in the Department of Biomorphology and Biotechnologies of the University of Messina, Italy, during the 2009 spring and fall seasons. Animal treatment and experimentation were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Four adult male light-adapted budgerigars M. undulatus (Shaw, 1805) (Psittaciformes, Psittacinae) were provided by a local dealer. The animals were acclimated for at least two days before the euthanasia to recover from the stress induced by transportation and the changes in environment. They were housed in individual cages under a L:D regimen of 12 hrs each with water and diet ad libitum. The budgerigars were euthanized by CO2 inhalation followed by cervical 541 dislocation and the eyes were immediately enucleated and processed for light and electron microscopy. Light Microscopy Four right eyeballs were cut with a razor blade along the equator and the posterior parts were ﬁxed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) at þ4 C for 2 hrs, washed with 0.2 M phosphate buffer (pH 7.4), and postﬁxed in 1% OsO4 in 0.2 M phosphate buffer (pH 7.4) at þ4 C for 1 hr. After dehydration in graded ethanol and acetone, the pectineal regions were isolated and ﬂat embedded in Durcupan. Semithin sections (1 lm) were cut with a LKB Ultrotome V ultramicrotome, stained with 1% toluidine blue in 1% borax and 1% pironine (Holstein and Wulfhekel, 1971) and viewed and photographed with a Zeiss Primo Star microscope. Transmission Electron Microscopy From the same specimens used for light microscopy (LM), ultrathin sections of silver interference-color were cut with a diamond knife on a LKB Ultrotome V ultramicrotome and collected on uncoated 200–300-mesh copper grids. Sections were stained with methanolic uranyl acetate and lead citrate (Reynolds, 1963). Micrographs were taken with a Philips CD-10 electron microscope at 80 kV. Scanning Electron Microscopy Four left intact eyeballs were ﬁxed as above indicated for light and transmission electron microscopy (TEM), dehydrated in graded ethanol and amylacetate, and critical point dried in CO2. The eyeballs were cut with a razor blade along equator and the posterior parts were mounted on aluminum stubs, coated with gold and examined and photographed with a Hitachi S-800 ﬁeld emission scanning electron microscope. Morphometric Analysis All micrographs used for the morphometric analysis were taken from the three parts of the pecten (base, folds, and bridge). The following four parameters were considered: mean external diameter of the capillaries, mean length of the luminal and abluminal microfolds (AM), mean number, and mean area of the pigment granules. All data were obtained from 15 semithin or ultrathin sections per animal (ﬁve for each part of the pecten for a total of 60 sections), collecting one semithin section every 100. For the mean external diameter of the capillaries and the mean number of pigment granules/unit area (UA) (2,500 lm2), the sections were observed with a Zeiss Primo Star microscope with a 40 objective and the images were captured using a Canon A620 Powershot camera and saved as tagged image format ﬁles with the Adobe Photoshop CS software. All micrographs were printed at the same ﬁnal magniﬁcation of 1,000 and were blindly assessed by three observers independently (Pasquale Aragona, Domenico Puzzolo, Antonio Micali): the mean and the standard deviation of the results were recorded. The mean diameter of the capillaries was calculated only from the blood vessels which showed a 542 MICALI ET AL. Fig. 1. Scanning electron micrograph of the pecten oculi of the budgerigar M. undulatus illustrating the different parts of the organ: base (B), folds (F), and bridge (Br). circular proﬁle. A Peak Scale Loupe 7x (GWJ Company, Hacienda Heights) micrometer was used as a scale calibration standard to calculate the diameters. 120 capillaries (40 capillaries for each part of the pecten) were considered. The mean number of pigment granules was calculated by counting all pigment granules observed in one randomly chosen UA of 2,500 lm2; 120 UAs (40 UA for each part of the pecten) were considered. For the mean area of the pigment granules and the mean length of the luminal and AM, TEM negative ﬁlms (Kodak 4489, 8 10 cm) were obtained at the same voltage, exposure time, and magniﬁcation. The negative ﬁlms were acquired (ratio 1:1) with an Epson Perfection scanner and processed with a Macintosh MacBook using the Adobe Photoshop CS software. TEM negative ﬁlms were converted into positive images at the same ﬁnal magniﬁcation of 7,500; the OPTILAB (Graphtek) software was used for the morphometric analysis. The mean granular area was calculated as follows: the perimeter of each granule was traced using the OPTILAB software options, so that the included area was automatically calculated and the results, obtained in square pixels, were converted into lm2. Forty granules/part of the pecten (for a total of 120 granules) were measured. The mean length of the luminal and AM was obtained only from the capillaries provided of a circular proﬁle. The mean length was calculated with the OPTILAB software options by tracing a line along the course of each microfold; the results in linear pixels were automatically converted into lm. Sixty capillaries (20 capillaries for each part of the pecten) were considered: for each capillary, 10 luminal and 10 AM were measured for a total 1,200 microfolds. All data were expressed in lm for linear values and in lm2 for surface values. Statistical Analysis Statistical analysis of the results was performed using the Student T-test by the S.A.S./Sta 6.0.3 software. A P value of 0.05 was considered as statistically signiﬁcant. RESULTS Structural and Ultrastructural Data The pecten oculi of the budgerigar is placed in the postero-inferior wall of the eyeball, oriented cranio-caudally and postero-anteriorly, along the course of the fetal ﬁssure. It is 1.8–2.2-mm long, 0.6–1.0-mm high, and 0.12–0.22-mm thick: as the entire eyeball of the budgerigar has a diameter of 8–9 mm, the pecten extends for about one-fourth of the eye. It is formed by 10–12 thin and rectilinear folds or pleats, uniform in their external morphology along the entire pecten. The folds are held together in their apical part by the transverse bridge, so that three different parts can be described: the base, the folds, and the bridge (Fig. 1). When viewed with the scanning electron microscopy (SEM) (Fig. 2a), each fold originates from the retinal surface with a thin part, the base, and then enlarges progressively and its surface becomes round and irregular, because of the presence of the pectineal capillaries of the folds. A transverse section of the basal part of the pecten (Fig. 2b) shows that it corresponds to the remnants of the fetal ﬁssure and is placed over the optic nerve ﬁbers. It consists of many capillaries and of two parallel vessels, an arteriole and a venule, whose caliber ranges between 60 and 80 lm. The wall of the basal arteriole is formed by endothelial cells, by a single or double layer of smooth muscle cells and by the basement membrane (Fig. 2c). The wall of the venule is formed by Fig. 2. Base region of the pecten oculi of the budgerigar M. undulatus. (a) Each fold (F) arises from the base (B). (b) The pecten merges along the remnants of the fetal ﬁssure (arrowhead); a pectineal arteriole (A) and a venule (V), collecting some capillaries (arrows), are evident. F ¼ fold; ONF ¼ optic nerve ﬁbers. (c) The wall of a basal arteriole is formed by endothelial cells (E), by smooth muscle cells (SMC) and by the basement membrane (arrow). (d) The wall of a basal venule is formed by endothelial cells (E) and by the basement membrane (arrow). ONF ¼ optic nerve ﬁbers. 544 MICALI ET AL. Fig. 3. Fold region of the pecten oculi of the budgerigar M. undulatus. (a) The capillaries (C) show a thick wall formed by endothelial cells (arrow). Pc ¼ pigment cell; Hy ¼ hyalocyte. (b) The tortuous course and the uniform caliber of the superﬁcial capillaries (arrow) is evident with the SEM. (c) A superﬁcial capillary shows endothelial microfolds on both luminal (arrows) and abluminal (double arrows) sides. The endothelial cells (inset) are connected by tight junctions (arrowhead). BM ¼ basement membrane; ILM ¼ inner limiting membrane; Hy ¼ hyalocyte with cytoplasmic processes (*); RBC ¼ red blood cell. (d) The cytoplasm (C) of an endothelial cell is reduced to a thin strip; rectilinear luminal microfolds (LM) and tortuous AM are present. THE PECTEN OCULI OF THE PARROT Fig. 4. Fold region of the pecten oculi of the budgerigar M. undulatus. (a) The wall of a superﬁcial capillary is formed by an endothelial cell provided of both luminal and AM (E1) and by two endothelial cells with ﬂat surfaces (E2). Arrows ¼ intercellular junctions; BM ¼ thick basement membrane; ILM ¼ inner limiting membrane; P ¼ pericytic processes. (b) Three interstitial pigment cells (Pc1-Pc2-Pc3), among which wide intercellular spaces (*) can be observed. C ¼ capillaries. 545 (c) In the cytoplasm of a pigment cell large granules (PG) and mitochondria (m) are present. (d) On the external surface of a capillary (C), a hyalocyte (Hy) is placed under the inner limiting membrane (ILM). P ¼ pericyte. (e) An isolated hyalocyte (arrow) is placed on the fold surface where the inner limiting membrane is interrupted. At higher magniﬁcation (inset), the hyalocyte (Hy) shows many processes (arrowhead). 546 MICALI ET AL. Fig. 5. Bridge region of the pecten oculi of the budgerigar M. undulatus. (a) The bridge (Br) is thinner on the optic nerve side, where the folds show an oblique course (*), and wider on the ciliary side, where the folds show an orthogonal course (**). (b) The bridge is formed by capillaries (C) and by pigment cells (arrows). (c) Interstitial pigment cells (Pc1-Pc2-Pc3), in whose cytoplasm large granules (PG) and mitochondria (m) are present. C ¼ capillary; * ¼ intercellular spaces. THE PECTEN OCULI OF THE PARROT 547 Fig. 6. Mean external diameter of the capillaries (expressed in lm) in the different parts of the pecten oculi of the budgerigar M. undulatus. No statistically signiﬁcant differences were found among the three parts of the organ. a continuous layer of endothelial cells and by the basement membrane: a layer of collagen ﬁbers (1–1.5 lm thick), probably derived from the mesenchyme of the fetal ﬁssure and exclusively placed at this site, separates the venular wall from the optic nerve ﬁbers (Fig. 2d). The transverse section of a fold (Fig. 3a) demonstrates the presence of many capillaries with a thick wall (up to 3 lm) formed by endothelial cells. Among the capillaries many pigment cells ﬁlled with dark granules can be observed. On the surface of each fold isolated hyalocytes are also present. The pectineal capillaries, when viewed with the SEM (Fig. 3b), show a uniform size and a tortuous course, so that each fold shows an irregular surface. With the TEM (Fig. 3c), the capillaries of the pecten are formed by endothelial cells extremely folded on both their luminal and abluminal sides and connected by tight junctions (Fig. 3c, inset). Endothelial cells rest on a basement membrane, 0.15–0.25-lm thick, which, in the superﬁcial vessels, is in close contact either with the long and irregular cytoplasmic projections of isolated hyalocytes or with the inner limiting membrane. At higher magniﬁcation (Fig. 3d), the endothelial cytoplasm is very thin (0.2–0.3 lm) and the microfolds are long and rectilinear on the luminal side and tortuous and shorter on the abluminal side. On the external surface of the folds, occasional capillaries with endothelial cells that show either both luminal and AM or smooth surface are evident. The cells are connected by tight junctions and rest on a lamellar and thicker (0.5–0.9 lm) basement membrane, on which some processes of pericytes adhere (Fig. 4a). Among the capillaries interstitial pigment cells (Fig. 4b) delimit wide intercommunicating intercellular spaces (0.5–1.5 lm), partially ﬁlled by thin processes. Their cytoplasm is ﬁlled with round pigment granules and large mitochondria (Fig. 4c). On the vitreal surface of the pecten, between the endothelial basement membrane and the inner limiting Fig. 7. Mean length (expressed in lm) of the luminal and abluminal processes of the capillaries in the different parts of the pecten oculi of the budgerigar M. undulatus. All measurements were statistically signiﬁcantly different from the others. membrane, large (up to 15 lm in diameter) hyalocytes are present (Fig. 4d): they show elliptical nuclei with dispersed chromatin, a thin halo of cytoplasm and many cellular processes. When the inner limiting membrane lacks owing to its loose adherence to the pectineal surface, long (up to 5–7 lm) rectilinear processes adherent to the pectineal capillaries surface can be observed with the SEM, either at low (Fig. 4e) or at higher magniﬁcation (Fig. 4e, inset). The bridge of the pecten (Fig. 5a) gathers the apical ends of each fold and shows different morphological patterns in its various parts. In fact, the folds originating from the optic nerve region are bent ciliarly and seem to be continuous with the bridge, with only a little change on their major axis. Near the ciliary extremity, the folds are bent on their major axis, thus penetrating the bridge with an angle of nearly 90 . When considered as a whole, the folds form an angle of 120–130 , opened toward the outer part of the eyecup. If viewed from above, the bridge has a triangular shape, being thinner on the optic nerve side and wider on the ciliary side. A transverse section of the bridge (Fig. 5b) shows many capillaries and a large number of pigment cells ﬁlled with dark granules. With the TEM (Fig. 5c) the capillaries show thick walls and a thin basement membrane, whilst the interstitial pigment cells are provided of many round and electron dense granules, of mitochondria and of many thin processes which jut into the wide (0.5–1.3 lm) intercellular spaces. 548 MICALI ET AL. Fig. 8. Mean number of the pigment granules/UA (2,500 lm2) in the different parts of the pecten oculi of the budgerigar M. undulatus. A statistically signiﬁcant difference was found among the three parts of the organ. Fig. 9. Mean area of the pigment granules (expressed in lm2) in the different parts of the pecten oculi of the budgerigar M. undulatus. A statistically signiﬁcant difference was found among all parts of the organ. Morphometric Analysis The morphometric analysis carried out on the mean diameter of the capillaries reveals no statistically signiﬁcant difference among the parts (Fig. 6). The evaluation of the mean length of the luminal and the AM of the endothelial cells demonstrates the highest values from those present in the folds region (1.6 0.4 lm and 1.2 0.2 lm, respectively), intermediate values from those present in the bridge (1.3 0.2 lm and 0.9 0.2 lm, respectively), and the lowest from those present in the base (1.1 0.3 lm and 0.8 0.1 lm, respectively). Statistically signiﬁcant differences are present among all considered groups (Fig. 7). The mean number of the pigment granules is particularly low in the base (11.9 1.3) and higher in the folds and in the bridge (62 4.7 and 68.6 2.9, respectively). However, the three parts of the organ show a statistically signiﬁcant difference for the number of granules (Fig. 8). As to the mean area of the pigment granules, the highest value is found in the bridge (1.4 0.3 lm2), whereas lower values (0.6 0.3 lm2 and 0.8 0.1 lm2, respectively) are observed in the base and in the folds. Statistically signiﬁcant differences are demonstrated among all groups (Fig. 9). DISCUSSION The pecten oculi is found in the vitreous chamber of the eye of all avians (Rochon-Duvigneaud, 1943) and it is considered an indirect retinal trophic system (Michaelson, 1954; Puzzolo, 1994), more effectively functioning during saccadic oscillations (Pettigrew et al., 1990). It is composed of three different parts: the base, originating from the optic nerve head, the folds, arranged like an inverted fan, and the bridge, running like a handrail along the vitreous margin of the organ. The base plays a relevant mechanic role, as it provides strong insertion of the pecten on the adjacent ocular layers along a zigzag line (Puzzolo et al., 1985b). This arrangement seems to be more functional than a rectilinear one in increasing its mechanical stability and its ability to withstand the inertial forces of the adjacent vitreous body (Tucker, 1975). Furthermore, it represents the site where the larger vessels (arterioles and venules) are found (Hossler and Olson, 1984). In the budgerigar these vessels are placed along the basal part, close to the optic nerve ﬁbers, so that the pecten, differently from other avians (Kiama et al., 1994; Braekevelt, 1998; Rahman et al., 2010), is composed only by capillaries. As to the folds, a relationship was proposed between the number of the pleats and the circadian activity and/ or the visual requirements of the single species (Braekevelt, 1998). In fact, a large and complicated pecten with 15–20 pleats is generally observed in photically active and visually oriented avians, whereas a pecten provided of smaller size and 4–5 pleats is found in avians with crepuscular or nocturnal habits and with reduced visual acuity. In the diurnal and visually oriented budgerigar, an intermediate value of 10–12 pleats, similar to the mallard (Braekevelt, 1990), was found. The bridge has been described as a relatively thick, pigmented, and poorly vascular plate (Tucker, 1975) with just a mechanic role. In fact, a ﬁrm connection between the vitreous and the pecten is generally ensured by vitreo-capsular ﬁbers which, in the chicken, penetrate into the superﬁcial microfolds of the pigment cells (Fischlschweiger and O’Rahilly, 1968) in a sawteeth fashion (Puzzolo et al., 1985b). In the bridge of the budgerigar no vitreal ﬁbers are observed, so that a THE PECTEN OCULI OF THE PARROT mechanical role cannot be conﬁrmed. On the contrary, different from all the birds examined, a large number of capillaries with thick wall are present, so that we can suggest a relevant trophic role also for this part of the pecten. The main structural components common to the different parts of the pecten are the hyalocytes, the pigment cells and the blood vessels. The hyalocytes (Seaman and Storm, 1965; Puzzolo et al., 1980; Ogawa, 2002) or peripectinate cells (Fischlschweiger and O’Rahilly, 1966; Uehara et al., 1990; Liebner et al., 1997) are placed on the vitreal surface of the pecten. They are considered as a subtype of blood-borne macrophages (Llombart et al., 2009) originated during embryogenesis from the primitive arteria cupulae opticae (Liebner et al., 1997). In the budgerigar, the hyalocytes are large cells placed under the pectineal limiting membrane, continuous with the retinal limiting membrane. Their position external to the vitreous body and adherent to the pectineal surface can be the consequence of a migration from the primitive vessels during the development. As to their distribution and number, we are not able to demonstrate the large number of isolated or even clustered hyalocytes observed in the adult chick (Uehara et al., 1996). It appears that, at least in the budgerigar, the hyalocytes are generally isolated and few in their absolute number. The pigment cells, derived from the outer leaﬂet of the optic cup, are considered as glial cells with unique morphological characteristics either during embryonic development or in the adult. During embryogenesis, a glial epithelium with well-evident tight junctions forms a primitive, but functional, glial blood–brain barrier (Gerhardt et al., 1996; Liebner et al., 1997). In the adult, the barrier function is lost (Reichenbach and Wolburg, 2005). However, the pecten glial pigment cells play other important roles: the absorption of the sunlight (Rahman et al., 2010), the regulation of the temperature in the eye (Bawa and YashRoy, 1974), the ﬂow of oxygen and carbon dioxide between the capillaries and the vitreous body (Jasinski, 1973), and the detoxiﬁcation of the retina by transforming vitreal ammonia into glutamine due to a strong expression of glutamine synthetase (Gerhardt et al., 1999). More recently, it was proposed that, in avians living in particularly difﬁcult environmental conditions, the pigment cells of the pecten may support brain function through a limited but critical melanin-initiated conversion of light to metabolic energy. Therefore, a direct correlation between the degree of melanization of the pecten and the ﬂight performances of the animal was hypothesized (Goodman and Bercovich, 2008). In the budgerigar, the morphological analysis shows a great number of pigment cells rich in mitochondria and a large amount of intercellular spaces (Schreck and Bowers, 1989). The morphometric analysis provides a strong support to the ultrastructural data as it demonstrates that the cellular area occupied by the granular melanin is statistically signiﬁcantly higher in the bridge region, thus suggesting a great involvement of this part of the organ in the metabolic activity of the avian eye. It has been shown that the pectineal vessels are formed by lymphatic vessels (Scala et al., 2002; Corona et al., 2004) and by blood capillaries (Braekevelt, 1988). In fact, in the pecten of Anas plathyrhynchos (Scala et al., 2002) lymphatic vessels, which might drain retinal 549 catabolites from the vitreous body (Corona et al., 2004), are demonstrated with the SEM analysis of vascular casts. In our study, we are able to identify only the presence of large intercellular spaces, whereas no lymphatic vessels are evident in the budgerigar. As to the blood capillaries, they are characterized by a maximum increase of the surface of the endothelial cells at the minimum thickness of their walls (Jasinski, 1973). In fact, even if apparently thick with the LM, they are formed by a thin strip of cytoplasm and, on both the luminal and abluminal surfaces, by a large number of microfolds (Braekevelt, 1988; Kiama et al., 1998) showing a great variability in number, length, and organization with the TEM. In the budgerigar, pleated membranes are present on both luminal and abluminal surfaces nearly in all endothelial cells; the luminal microfolds are generally rectilinear, whereas the AM are always tortuous. The presence of the microfolds determines an enormous enlargement of the inner and outer surfaces of the vessels. The demonstration of carbonic anhydrase activity in the apical and basal microfolds of the pectineal endothelial cells (Eichhorm and Flugel, 1988) indicates that the pecten may play a signiﬁcant role in the intraocular pH regulation, as already suggested by Brach (1977) after experimental destruction of the pecten and by Gerhardt et al. (1999) during its development. Furthermore, as it was shown that the pectineal endothelial cells possess an extraordinarily high amount of glucose transporter isoform-1 (Gerhardt et al., 1999), it is possible to propose that in the budgerigar the folds region, characterized by the longest luminal and AM, may have a trophic role as a glucose provider to fuel the glycolysis of the avascular retina (Wolburg et al., 1999). Conversely, the bridge region, where the number and the area of the pigment granules show the highest values, seems to be more involved in the production of energy. This study shows that morphometric analysis, used together with morphological features, allows a more precise description of a complex structure such as the pecten oculi in the budgerigar, giving hints about the possible functional roles of the different parts of the organ. ACKNOWLEDGEMENTS The authors thank Mr. Sebastiano Brunetto of the Department of Biomorphology and Biotechnologies (University of Messina, Italy) for the technical assistance. All the authors declare no commercial relationships relevant to the subject matter of the article. LITERATURE CITED Bawa SR, YashRoy RC. 1972. Effect of dark and light adaptation on the retina and pecten of chicken. Exp Eye Res 13:92–97. Bawa SR, YashRoy RC. 1974. Structure and function of vulture pecten. Acta Anat 89:473–480. Brach V. 1977. The functional signiﬁcance of the avian pecten: a review. Condor 79:321–327. Braekevelt CR. 1984. Electron microscopic observations on the pecten of the nighthawk (Chordeiles minor). Ophthalmologica 189:211–220. Braekevelt CR. 1988. Fine structure of the pecten of the pigeon (Columba livia). Ophthalmologica 196:151–159. 550 MICALI ET AL. Braekevelt CR. 1990. Fine structure of the pecten oculi of the mallard (Anas plathyrhynchos). Can J Zool 68:427–432. Braekevelt CR. 1994. Fine structure of the pecten oculi in the American crow (Corvus brachyrhynchos). Anat Histol Embryol 23:357–366. Braekevelt CR. 1998. Fine structure of the pecten oculi of the emu (Dromaius novaehollandiae). Tissue Cell 30:157–165. Braekevelt CR, Richardson KC. 1996. Fine structure of the pecten oculi in the Australian galah (Eolophus roseicapillus) (Aves). Histol Histopathol 11:565–571. Corona M, Scala G, Perrella A. 2004. Angioarchitecture of the duck pecten. Biomed Res 15:19–25. Dieterich CE, Dieterich HJ, Spycher MA, Pfautsch M. 1973. Fine structural observations of the pecten oculi capillaries of the chicken. Freeze-etching, scanning and transmission electron microscopic investigations. Z Zellforsch 146:473–489. Dieterich HJ, Dieterich CE. 1974. Licht- und elektronenmikroskopische Untersuchungen am Pecten oculi verschiedener Vogelarten. Verh Anat Ges 68:476–478. Eichhorm M, Flugel C. 1988. Histochemical demonstration of carbonic anhydrase and Naþ/Kþ-ATPase in the pecten oculi of the fowl. Exp Eye Res 47:147–153. Fielding M. 1972. The ultrastructure of the pecten oculi in the domestic fowl. J Anat 113:295–297. Fischlschweiger W, O’Rahilly R. 1966. The ultrastructure of the pecten oculi in the chick. Acta Anat 65:561–578. Fischlschweiger W, O’Rahilly R. 1968. The ultrastructure of the pecten oculi in the chick. II. Observations on the bridge and its relation to the vitreous body. Z Zellforsch 92:313–324. Gerhardt H, Liebner S, Wolburg H. 1996. The pecten oculi of the chicken as a new in vivo model of the blood-brain barrier. Cell Tissue Res 285:91–100. Gerhardt H, Schuck J, Wolburg H. 1999. Differentiation of a unique macroglial cell type in the pecten oculi of the chicken. Glia 28:201–214. Goodman G, Bercovich D. 2008. Melanin directly converts light for vertebrate metabolic use: heuristic thoughts on birds, Icarus and dark human skin. Med Hypotheses 71:190–202. Gültiken ME, Yildiz D, Onuk B, Karayigit MO. 2011. The morphology of the pecten oculi in the common buzzard (Buteo buteo). Vet Ophthalmol, doi: 10.1111/j.1463-5224.2011.00965.x. Holstein AF, Wulfhekel V. 1971. Die semidünnschnitt-tecknik als Grundlage für eine cytologische Beurteilung der Spermatogenese des Menschen. Andrologie 5:65–69. Hossler FE, Olson KR. 1984. Microvasculature of the avian eye: studies on the eye of the duckling with microcorrosion casting, scanning electron microscopy, and stereology. Am J Anat 170:205–221. Jasinski A. 1973. Fine structure of capillaries in the pecten oculi of the sparrow, Passer domesticus. Z Zellforsch 146:281–292. Kiama SG, Bhattacharjee J, Maina JN, Weyrauch KD. 1994. A scanning electron microscope study of the pecten oculi of the black kite (Milvus migrans): possible involvement of melanosomes in protecting the pecten against damage by ultraviolet light. J Anat 185:637–642. Kiama SG, Bhattacharjee J, Maina JN, Weyrauch KD. 1997. Surface specialization of the capillary endothelium in the pecten oculi of the chicken, and their overt roles in pectineal haemodynamics and nutrient transfer to the inner neural retina. Acta Biol Hung 48:473–483. Kiama SG, Maina JN, Bhattacharjee J, Weyrauch KD, Gehr P. 1998. A scanning electron microscope study of the luminal surface specializations in the blood vessels of the pecten oculi in a diurnal bird, the black kite (Milvus migrans). Ann Anat 180:455–460. Kiama SG, Maina JN, Bhattacharjee J, Mwangi DK, Macharia RG, Weyrauch KD. 2006. The morphology of the pecten oculi of the ostrich, Struthio camelus. Ann Anat 188:519–528. Liebner S, Gerhardt H, Wolburg H. 1997. Maturation of the bloodretina barrier in the developing pecten oculi of the chicken. Dev Brain Res 100:205–219. Llombart C, Nacher V, Ramos D, Luppo M, Carretero A, Navarro M, Melgarejo V, Armengol C, Rodrı́guez-Baeza A, Mendes-Jorge L, Ruberte J. 2009. Morphological characterization of pecteneal hyalocytes in the developing quail retina. J Anat 215:280–291. Meyer DB. 1977. The avian eye. In: Crescitelli F editor. Handbook of sensory physiology Berlin: Springer, Vol. VIII/5. p 549–612. Micali A, Puzzolo D, Parducci F, Spatari G, Urbani P, La Fauci MA. 1988. Structural and ultrastructural investigations on the papillary cone in the eye of the gecko (Tarentola mauritanica). It J Anat Embryol 93:217–236. Michaelson IC. 1954. Retinal circulation in man and animals. Springﬁeld: CC Thomas. Ogawa K. 2002. Scanning electron microscopic study of hyalocytes in the guinea pig eye. Arch Histol Cytol 65:263–268. Parducci F, Micali A, La Fauci MA, Puzzolo D. 1987. Comparative morphogenesis of the pecten oculi in chick and pigeon embryos. It J Anat Embryol 92:145–158. Pettigrew JD, Wallman J, Wildsoet CF. 1990. Saccadic oscillations facilitate ocular perfusion from the avian pecten. Nature 343:362– 363. Puzzolo D. 1994. Morphological adaptation of the vertebrate eye to the environment. It J Anat Embryol 99:17–100. Puzzolo D, de Simone I, Farina F, La Fauci MA, Caminiti G. 1980. On the development of the pecten oculi in the chick embryo. A light and scanning electron microscopic study. It J Anat Embryol 85:105–116. Puzzolo D, La Fauci MA, Micali A, Pisani A, Cutroneo G, Arco A. 1985a. The pecten oculi of the pigeon. Scanning electron microscopic study. Riv Biol 78:269–281. Puzzolo D, Micali A, Pisani A, Arco A, Cutroneo G. 1985b. Scanning electron microscopic study on the relationship between the pecten oculi and the vitreous capsule in the eye of the adult chicken. Quad Anat Prat 61:93–98. Rahman ML, Lee E, Aoyama M, Sugita S. 2010. Light and electron microscopy study of the pecten oculi of the jungle crow (Corvus macrorhynchos). Okajimas Folia Anat Jpn 87:75–83. Raviola E, Raviola G. 1967. A light and electron microscopic study of the pecten of the pigeon eye. Am J Anat 120:427–461. Reichenbach A, Wolburg H. 2005. Astrocytes and ependima glia. In: Kettenmann H, Ramson BR, editors. Neuroglia. Oxford: University Press. p 19–35. Reynolds ES. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212. Rochon-Duvigneaud A. 1943. Les yeux et la vision des Vertébrés. Paris: Masson et Cie. Scala G, Corona M, Mirabella N, Perrella A, Pelagalli GV. 2002. Microvasculature of the pecten oculi in Anas plathyrhynchos. It J Anat Embryol 107:65–75. Schreck RE, Bowers RR. 1989. Effect of the barring gene on eye pigmentation in the fowl. Pigment Cell Res 2:191–201. Seaman AR, Storm HK. 1965. Electron microscope observations on the hyalocytes of the vitreous body in the domestic fowl (Gallus domesticus). Exp Eye Res 4:13–16. Smith BJ, Smith SA, Braekevelt CR. 1996. Fine structure of the pecten oculi of the barred owl (Strix varia). Histol Histopathol 11:89–96. Tucker R. 1975. The surface of the pecten oculi in the pigeon. Cell Tissue Res 157:457–465. Uehara M, Oomori S, Kitagawa H, Ueshima T. 1990. The development of the pecten oculi in the chick. Nippon Juigaku Zasshi 52:503–512. Uehara M, Imagawa T, Kitagawa H. 1996. Morphological studies of the hyalocytes in the chicken eye: scanning electron microscopy and inﬂammatory response after the intravitreous injection of carbon particles. J Anat 188:661–669. Wolburg H, Liebner S, Reichenbach A, Gerhardt H. 1999. The pecten oculi of the chicken: a model system for vascular differentiation and barrier maturation. Int Rev Cytol 187:111–159.