histological analysis of eyeballs of the striped owl rhinoptynxclamator

Histological analysis of eyeballs of the striped owl

Rhinoptynxclamator

P. C. O. C. Jezler¹, M. B. P.Braga¹, E. Perlmann¹, R. Squarzoni¹, M. I. Borella²,P. S. M.

Barros¹, L.Milanelo³, and A. Antunes4*

1Laboratory of Experimental Comparative Ophthalmology, School of Veterinary Medicine, University of

Sao Paulo, Brazil 2Department of Histology and Embryology, Biomedicine Science Institute, University of Sao Paulo, Brazil 3Ecological Park of Tiete, Sao Paulo, SP, Brazil 4Institute of Physics, Federal University of Uberlandia, Av Joao Naves de Ávila, 2121 - Uberlandia MG * Corresponding author:e-mail: [email protected]

Keywords avian eye;ocular structure;owl;vision;pecten oculi

Birds, with some exceptions, are incredibly visual animals. Birds rely most heavily on their ability to assess their visual environment. High visual acuity is not only necessary to find and acquire food, but also to identify co specifics and potential mates, and to quickly identify and escape from predators. The owls are nocturnal birds that play an important role in the balance of ecosystem for preventing overpopulations of preys and eliminating faulty individuals. They have sharpened visual capacity and present complex retinal structure, interesting for research of the functioning of its visual system. In this chapter we presented the morphological and histologicalcharacterization of the eyeballs of the striped owl Rhinoptynxclamator, peculiar birds founded in Brazil.

Introduction

More so than any other terrestrial vertebrate, birds rely most heavily on their ability to assess their visual environment [1]. One important consequence of sunlight striking our planet has been the evolution of eyes. Scientists have always been fascinated by the design, function and adaptations of the eye to different habitats [2]. South America's largest country is also one of the richest for birds. Although the Brazilian bird fauna is one of the most diverse in the world,the Strigidae Family is distributed around all the continents with exception in Antarctica, having totalized 146 species in the World [3], of these 22 occurs in Brazil [4]. The Rhinoptynxclamator(Fig. 1)lives in South Americaextending itself for great part of the Tropical and Subtropical Zone, and parts of Central America [3, 5, 6]. Nocturnal habits are rare amongst the birds, with approximately 5% of the species. By having these habits, owls present special adaptations and morphologies to the nocturnal life, for example, the presence of the face record of penaltythatplays an important role of sonorous reflector and aquiet flightthat assists them in hunting. This species is associated with open environments, as urban and agricultural areas, fields and forest edges of Atlantic forest [3].

Fig. 1 Image of a young Rhinoptynx clamator

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The ear openings in the skin are slit-like and very large, extending from below the lower jaw up to the top of the head, thus occupying the entire height of the head. The preaural flaps overlap the ear openings, which therefore open towards the side of the head. The ear openings in the skin, the postaural skin folds and the preaural flaps are perfectly symmetrical between the left and the right side. It body anatomy include: length up to 37 cm, wingspan 22, 8 -29, 4 cm and weight320-546 g. It lives in open or semi-open grassland, small woods and bushes.They live in the maximum of 10-20 years [1]. Birds, withsome exceptions, are incredibly visual animals. The avian globe, in relation to the size of the skull, is very large, which is advantageous for species relying heavily on their ability to interpret and respond to their visual environment. Approximately 50% of the cranial volume of the skull is occupied by the eyes. The eyes of some owls and hawks are even having larger than human eyes. A larger eye allows a larger image to be projected into the retina, thus contributing vastly to the vision[1].

Ophthalmic Anatomy

Ocular anatomy of the vertebrate has been remarkably conserved throughout evolution [13]. The orbit is formed by the frontal, prefrontal, sphenoid, ethmoid, palatine, quadrate bones, and the jugular arch. The dorsal and temporal aspects of the globe are unprotected by the bony orbit, but the rest of the globe fits very snugly within the orbit [1]. The extraocular muscles consist of the medial, lateral, dorsal, and ventral rectus muscles, and the dorsal and ventral oblique muscles. The oblique and rectus muscles are thin and relatively poorly developed [1, 8]. The retractor bulbi muscle is absent in the avian species, and in its place are the quadrates and pyramidalis muscles. The pyramidalis muscle originates from the posterior pole sclera where it loops through a sling formed by the quadratus muscle; both are innervated by cranial nerve VI [8]. These muscles function to move the nictitating membrane. In owls a scleral sesamoid bone encloses the tendon of the pyramidalis muscle [1]. The globe is very large in relation to body size [8]. The anterior and posterior segments of the globe are united by a variably shaped intermediate region based on the scleralossicles [1]. The posterior segment is relatively much larger than the anterior segment [8]. In the temporal aspect, the asymmetry between the anterior and posterior segments of the globe is more extensive than the nasal aspect [1]. Three basic shapes are typical: a flat, globoseand tubular. In the owl the eye shape is tubular, in which the concave intermediate segment is elongated antero-posteriorly, forming a tube before joining the posterior segment at a sharp angle, and the cornea positioned anteriorly [1, 8]. Upper and lower eyelid and membrane nictitans are present. The lower lid is more mobile than the upper one [8]. Palpebral margins were darkly pigmented with a conspicuous thickening of the marginal tissue, particularly along the dorsal eyelid [9]. Eyelid movement is controlled by 4 striated muscles. The levatorpalpebraesuperioris muscle, which has both skeletal and smooth muscle fibers, elevates the upper eyelid, whereas the depressor palpebraeventralis muscle, as its name implies, depresses the lower lid [1]. The nictitating membrane lies in the dorsonasal quadrant of the conjuntival sac. The nictitating membrane is well developed, actively mobile, transparent, translucent, or opaque white, as in owls and some other avian species [1, 8]. Drawn from the nasal canthus, the nictitans is moved by contracture of the pyramidalis muscle [8]. The gland of the nictitating membrane lies in the inferior nasal quadrant of the orbit and secretes via its ducts into the conjunctival space between the bulbar surface of the nictitans and the cornea [1]. Meibomiam glands are also absent [1]. Owls may have lower volumes of aqueous tear production than other birds, as their orbital lacrimal glands are small or absent [9]. The lacrimal gland is located inferior temporal area to the globe. The Harderian gland is found adjacent to the posterior sclera, near the base of the nictitants but not part of it. Two lacrimal puncta drain lacrimal secretions into a nasolacrimal duct and then to the nasal cavity [1, 8].

Materials and methods

The eyeballs structures of the Rhinoptynxclamator were investigated. The owls are frequently taken to rehabilitation in the Ecological Park of Tiete, unfortunately due to healthy conditions some animals died in a short period of time. Immediately after death, the eyeballs were carefully enucleated, washed in saline solution and submerged in a sterile container with fixative solution, 10% formaldehyde, for approximately 24 hours. Tissues were first dehydrated through graded alcohols and cleared in xylol, and immersed in liquid paraffin at 60ºC. Subsequently obtaining the paraffin blocks, 5 µm thick sections were cut and stained with hematoxylin and eosin and examined by light microscopy.

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Fibrous Tunic (Sclera and Cornea)

The shape of the globe is formed andmaintained by hyaline cartilage (Fig. 2) in the sclera of the posterior segment and by 10 to 18 scleral ossicles in the sclera of the intermediate segment, but most avian species have 14 to 15 [1, 8]. The ossicles (Figs. 3 and 4) provide protection and shape theglobe, functioningas the site of origin for the striated ciliary muscles. These ossicles are located anteriorly underneath or external to the ciliary body from the limbus to the equatorial region of the globe [1]. Pectinateligaments join the limbus to the ciliary body and the iris. Aqueous humor is drained through the spaces between the pectinate ligaments, into the Schlemm’s canal [1].

The cornea, similar to other vertebrates, is transparent and avascular and clear with dense brown pigmentation at the limbus, and corresponds the anterior portion of the fibrous tunic of the globe [1, 8, 9]. Eagle or owl tubular eyes have a strongly curved cornea [10]. The translucency of the cornea enables it to perform two main functions, to refract light and to allow sufficient quantity and quality of light into the eye to form an image on the retina [10]. The cornea consists of the corneal epithelium, superficial anterior limiting lamina (Bowman’s layer), stromal, Descemet’s membrane, and endothelium [1, 10].

hc

os

hc

Fig.3 The sclera is reinforced by overlapping ossicles (os)and elsewhere by hyaline cartilage (hc)(hematoxylin-eosin, original magnification X40).

Fig.4 The sclera and sclera ossicles and hyaline cartilage(hematoxylin-eosin, original magnification X10).

Fig.2 Hyaline cartilage (hc), scleral ossicles (so) and choroid (co)(hematoxylin-eosin, original magnificationX40).

hc

so

co

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The epithelial and endothelial layers are responsible to keep the cornea desiderated for of the cornea by acting as a hydrophobic barrier and pump [1]. The corneal endothelium functions as a physiologic pump to remove and transport fluid into the anterior chamber and regulates hydration of the corneal stromal collagen matrix, which provides mechanical strength [10].

Vascular Tunic (Iris, Ciliary body, Choroid)

The iris, ciliary body, and choroid make up the vascular tunic or uvea of the globe [1]. The anterior uvea refers to the iris and the ciliary body. The ciliary body is contiguous with the choroid at its posterior aspect [13]. The iris controls the amount of light that entersthe eye, and the ciliary body alters the focal power of the lens, produces aqueous humor that supplies nutrition to ocular structures, and aids in regulating intraocular pressure (IOP) [13]. The iris consists of numerous blood vessels, fibroblasts, nerves, collagen, epithelial cells, and an extensive musculature [1]. The iris contains striated sphincter,dilator muscles,myoepithelium and smooth muscle [8]. Iridal color results from the amount and type of pigmentation, and the degree of vascularization [1]. Alterations in the size and shape of the avian pupil can be quite extensive and much faster in avian species than in mammals [1]. Circumferentially, the contractile components that are the principal constrictors of the iris consist of a peripupillarymyoepithelium, an annular band of smooth muscle that provides sustained papillary miosis, and a broader band of striated muscle, which is the principal constrictor of the iris [1]. The radial contractile facilitate dilation of the iris. The consensual papillary light reflex is absent because of complete decussation of the nerve fibers at the optic chiasm [1]. The iris in normal striped owls was brown with variable amounts of brown pigment flecks and fine vasculature present in the stroma [8]. This angle is well developed and drained by two annular channels [8]. The pupils of most birds aresymmetrical and mid-range in room light [9]. The ciliary body is responsible for aqueous by the ciliary body, the generation of intraocular , muscular influence of conventional aqueous humor outflow and lens accommodation [1, 11]. It suspends the lens by the zonular fibers and also forms the ciliary processes by the action of the anterior e posterior sclerocorneal muscles. The anterior sclerocorneal muscles are the largest in owls [1]. The ciliary muscles are also striated in avian species, allowing quick accommodation. Three muscles are involved in accommodation. The muscle of Crampton,the most anterior ciliary muscle, is responsible for the change in the corneal curvature. Brücke’s muscle and Müller’s muscle are posterior to the muscle of Crampton [14].

Contraction of theses muscles exerts force on the ciliary processes (Figs. 5 and 6) that are fused to the lens capsule equator. The avian eye has an accommodation range from 2 to 50 D, using these three mechanisms [14]. The choroid consists of mainly thin-walled blood vessels and pigmented connective tissue [1]. It is the main source of nutrition for the outer layers of the retina, which are immediately adjacent [12]. The choroid can be divided externally to internally into 5 layers [12]. The first layer is the suprachoroidea. This layer is avascular, pigmented connective tissue that forms a transition between the sclera and the choroid. In birds, this region also contains a layer of nonvascular smooth muscle cells. The function of this layer of smooth muscle within the suprachoroidea is presently unknown, but its location suggests that

Fig.5 CP, ciliary processes(hematoxylin-eosin, original magnification X10).

Fig. 6 CP, ciliary processes(hematoxylin-eosin, original magnification X40).

cp

cp

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it could play a significant role in uveoescleral aqueous humor outflow and IOP regulation [12]. The second layer is the spatiumperichoroideale, consists of striated and nonstriated muscle fiber bundles and cords of connective tissue [1]. These vessels serve as a large reservoir that may assist in IOP regulation by removal of fluid from the blood vessels [12]. The third layer is the lamina vasculosa and consists of predominately arteries. The lamina choriocapillaris is the fourth layer and helps provide oxygen to the retina and the lamina basalis; it is a thin basement membrane that separates the choriocapillaris from the retina pigmented epithelium [1]. The tapetal layer varies among species, and it is absent in the pig, squirrel, and many nonhuman primates [12].

Nervous Tunic

Retina

The retinaa direct extension of the central nervous system is the visual sensory component of the eye and consists of a layer of pigmented epithelium and a neurosensory layer. When present, the retinal circulation supplies nutrients to the neural layers of the retina while the photoreceptors are maintained by the choroidal circulation. The supplemental include the choroid body, the lentiform body and the falciform process of teleost, the pecten oculi of birds [2]. Classically, 10 layers (Figs. 7 and 8)are described in retinal histology [12].

The retina layer of birds is the same as those of other vertebrates; however, some variations exist in morphology, areas of visual acuity, and retinal vascularization [1]. The retina is avascular, atapetal, anangiotic with a large, pigmented, vascular pecten extending into the vitreous [14]. Choroid and pectenare well-developed [1]. This high metabolic activity of the retina demands an efficient system of delivery of the nutrients required and removal of the by-products of metabolism. The retina contains photoreceptors, rods and cones [1].

Fig.7 The avian retina is a relatively thicker, avascular structure in which the layers are more clearly distinguishable(hematoxylin-eosin, original magnification X40).

1 Retinal pigment epithelium

2 Photoreceptor layer

3 External limiting membrane

4 Outer nuclear layer

5 Outer plexiformlayer

6 Inner nuclear layer

7 Inner plexiformlayer

8 Ganglion cell layer

9 Optic nerve fiber layer

10 Internal limiting membrane

1 2 3 4 5 6 7 8 9 10

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Rod photoreceptors contain the photopigment rhodopsin. They respond to most wavelengths of light in the visible spectrum and respond to light of lower luminance or brightness. Cones function in sharp visual acuity, color, and daylight vision. They arepresent within the retina as a single cell or in a pair. The double cone is the dominant photoreceptor in diurnal animals [1]. The retinas of most bird species also contain multiple spectral classes of cones, and spectral sensitivity maxima in the approximate region of red, green, and blue are common. The oil droplet, also present within the single cones, provides light filtration, allowing certain wavelengths of light to be transmitted to the outer segments of the photoreceptors, and it aids in the detection of retinal polarization [1]. The detection of shapes, motion, and low light vision is provided by the rod photoreceptors. Some avian species have 1 or 2 cone-rich, rod-free regions within their retina. These areas are called foveas, and are used to perceive hues of color, high resolution, and binocular fixation. Fovea are depressions within the retina that contain only cones and in which images are focused for increased visual acuity [1]. Some species are afoveate, others are monofoveate, and some are bifoveate [8]. Owls have only temporal foveae, whereas condors and black vultures have only a nasal fovea. In owls this is probably due to their more frontally placed eyes and greater binocular field, which helps summate light from both eyes under the dim lighting conditions in which they are most active [1]. The pecten oculi is a structure peculiar to the avian eye [2]. The pecten is a nonsensory, highly vascular pigmented structure of greatly variable size extending the optic nerve into the vitreous chamber [1]. The pecten is situated in the lower posterior temporal quadrant of the fundus [2]. Three morphological types of pecten oculi are recognized: conical type, vaned type and pleated type [8]. The shapes and number of pleats of the pecten vary among nocturnal and diurnal birds (Figs.9 and 10). Raptors, and most other birds, have the pleated type of pecten. In general, diurnal birds have a larger pecten with more folds than the nocturnal species [1].

Fig.8 Histological cut the avian retina the Rhinoptynxclamator(hematoxylin-eosin, original magnification X40).

Fig.9 The avian pectin(hematoxylin-eosin, original magnification X40).

Fig.10 The base of the pecten as it internally lines the nerve fibers that form the optic nerve(hematoxylin-eosin, original magnification X10).

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The pecten must not be too large to interfere with the optical function of the eye and must also be stable enough to resist lateral swing. It overlies the optic disc and the apex projects into the vitreous humour in form of a cone [2].Since the optic disc constitutes a blind spot on the retina, it is of great advantage that the pecten is so located to optimize the extent of the retinal available for image capture [2]. The pecten probably has a primary nutritive function but has been credited with over 30 possible functions [1]. These include intraocular pressure regulation, intraocularpH regulation, stabilization of the vitreous, reduction of intraocular glare, serving as a blood and fluid barrier for the retina and vitreous body, and above all as a supplemental nutritive organ [15]. The pecten also provides an oxygen gradient to the retina and maintains a constant intraocular temperature [1]. Melanin is present in abundance at the apical and peripheral pecten. These pleomorphic melanocytes form incomplete sheaths along the vast plexus of capillaries. They provide some structural support to the pecten and protect the blood vessels from ultraviolet light and oxygen radicals [1]. Pigment epithelial processes contain pigment granules and respond to light by elongation between rods. The fundus appearance is a gray or red background speckled with heterogeneous pigmentation through which choroidal vessels may be seen in some species [8]. The optic nerve is most highly developed in falconiformes and is less developed in nocturnal avian species [1].

The Lens

The avian lens (Fig. 11) differs greatly from that of mammals in that it has an annular pad around its central [1]. An equatorial annular pad formed of modified lens fibers is present and may be very prominent. Between the central body of the lens and the annular pad is a fluid-filled cleft or lenticular space [8]. The lens is a soft, pliable, transparent, avascular, biconvex body with an anterior surface that is flatter or less curved than the posterior surfaces and of variable shape. The shape lens is spherical in nocturnal species and flattened anteriorly in some diurnal species [8, 17]. The basic function of the avian lens, along with the cornea, serves to refract light onto the retina, thus producing a clear, sharp image [1, 12]. The lens is held in place by the vitreous, zonular fibers that bind the ciliary process to the annular pad at the lenticular equator, and by the support of the iris [1]. The nutrition support of the lens is provided by the aqueous humor [1].

The lens is high in protein (35%) and water (65%), its relative dehydration and its sodium and potassium gradients are maintained by an active sodium-potassium adenosine triphosphatase pump in the anterior lens epithelium [1]. Diurnal species of birds have a flatter anterior lens surface compared with nocturnal and aquatic species. The avian lens capsule is relatively thin and consists of type IV collagen. Around the main lens body is the annular pad, which consists of radially aligned large lens fibers that are capable of withstanding the applied pressure during accommodation [1].

Fig.11 Histological cut the avianlens(hematoxylin-eosin, original magnification X40).

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Accommodation in birds involves changes in corneal curvature, anterior movement of the lens, and lens deformation [8]. Lens power is increased by contraction of the striated meridionalciliary muscles [8]. The Brucke’s muscle posteriorly and Crampton’s muscle anteriorly move the ciliary body axially, compressing the lens by exerting pressure on the annular pad [8]. Using these mechanisms, the avian eye has an accommodation range from 2 to 50 D [14].

Concluding Remarks

The chapter presents a brief description about manipulation and preparation of ocular tissue and images of some structures that can be perfectly studied using histological analysis. It was elaborated to identify the anatomicstructures of the owl’s eyes understanding that this information can be important to compare with others avian species eyes. Further studies involving the role of vision in animal and human interrelating their differences and functions can be performed as well as their role in preventive studies.

Acknowledgements The authors thank the Ecological Park of Tiete for access to the owls.

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histological analysis of eyeballs of the striped owl rhinoptynxclamator

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