Comp Clin Pathol (2006) 15:169–174 DOI 10.1007/s00580-006-0628-0
ORIGINAL ARTICLE
Morphological, cytochemical, and ultrastructural observations on the blood cells of the reptile Tupinambis merianae (Squamata) R. L. Carvalho & M. M. Antoniazzi & C. Jared & A. M. J. Silva & A. A. Santos & M. I. Egami
Received: 20 March 2006 / Accepted: 13 June 2006 / Published online: 13 September 2006 # Springer-Verlag London Limited 2006
Abstract Tupinambis merianae is a lizard of the Brazilian fauna and belongs to the Squamata order. Blood cell data are scarce. Blood samples from six specimens of adult T. merianae were used to evaluate some hematological parameters. For structural analysis, 2 ml of blood was collected in the presence of EDTA. Part of the blood was used for preparing blood smears which were submitted to the methods of Leishman, Laür, and toluidine blue, and the cytochemical reactions of periodic acid-Schiff, sirius red, sudan black B, and ortho-toluidine-H2O2. The remainder was centrifuged and the leukocyte buffy coat was fixed in Karnovsky’s fluid for electron microscopy examination. The following blood cells were identified: mature and immature erythrocytes, spherical and elliptical thrombocytes, heterophilic granulocytes, eosinophils and basophils types I and II, lymphocytes, monocytes, and azurophilic cells. The more significant results obtained were: the presence of glycogen in the cytoplasm of the thrombocytes, heterophils, and basophils; the presence of basic polyaminoacid-rich proteins in the granules of heterophils and R. L. Carvalho (*) : M. I. Egami Departamento de Morfologia, UNIFESP/EPM, São Paulo, Brazil e-mail:
[email protected] M. M. Antoniazzi : C. Jared Laboratorio de Biologia Celular, Instituto Butantan, São Paulo, Brazil A. M. J. Silva Departamento de Parques e Áreas Verdes, Secretaria Municipal do Verde e Meio Ambiente do Estado de São Paulo, São Paulo, Brazil A. A. Santos Universidade Adventista de São Paulo, São Paulo, Brazil
eosinophils and myeloperoxidase in the granules of the heterophils, eosinophils, and azurophilic cells; and sudanophilic small granules in the heterophils, eosinophils, and azurophilic cells. More detailed morphological aspects of the cells were observed by means of ultrastructural analysis. Keywords Reptilia . Lizard . Tupinambis . Blood cells . Morphology . Cytochemistry
Introduction Tupinambis merianae is an omnivorous lizard of SouthAmerican herpetofauna with diurnal habits which lives in holes dug in the earth in forests, savannah (Cerrado), and semiarid regions (Caatinga), from the south of the Amazon to the north of Argentina. Data regarding the hematology of this species are scarce; even simple morphological descriptions of the cell types to be found in the peripheral blood have not been reported. Morphological work, using light and electron microscopy and cytochemistry, on blood cells of other reptiles served as a basis for the present study. Among these were papers on snakes (Egami and Sasso 1988; Dotson et al. 1995; Salakij et al. 2002), turtles (Daimon et al. 1987; Cannon 1992; Alleman et al. 1992; Knotková et al. 2002), lizards (Caxton-Martins 1977; Caxton-Martins and Nganwuchu 1978; Eliman 1997), and crocodilians (Caxton-Martins 1977; Mateo et al. 1984; Moura et al. 1997, 1999; Oliveira et al. 1997, 1998a,b). The aim of this work is to contribute to the knowledge concerning the general morphology of blood cells and the chemical nature of their cytoplasmic components and to establish a general hematological profile for the Squamata.
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Material and methods Blood samples from six adult T. merianae, regardless of sex, were examined. For structural analysis, 2.0 ml of peripheral blood from the tail vein was collected in the presence of 0.2 ml of 10% EDTA. Half of the blood volume was used in the preparation of glass-slide smears, which were submitted to the Leishman staining method for general morphological analysis, to the Laür method for characterizing of immature erythrocytes, and to toluidine blue for demonstration of metachromasy. The following cytochemical methods were also applied: periodic acidSchiff for glycogen detection, sirius red for basic poliaminoacids detection, sudan black B for membrane lipid detection, and ortho-toluidine-H2O2 for the detection of myeloperoxidase enzymes. The rest of the volume (around 1.0 ml) was centrifuged and the leukocyte buffy coat was fixed in a mixture of 5% glutaraldehyde and 4% paraformaldehyde in sodium cacodylate buffer 0.1 M, pH 7.2 (Karnovsky solution), postfixed in 1% osmium tetroxide, contrasted in 1% solution of uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections (60 nm) were cut using a Sorvall MT6000 ultramicrotome and examined in a LEO 906E transmission electron microscope, operating at 80 kV.
Results and discussion In the peripheral blood cells of T. merianae the following blood cells were identified: mature erythrocytes; reticulocytes; spherical and elliptical thrombocytes; heterophils with acidophilic and basophilic granules of two basic shapes (rod-shaped and spherical) and different sizes; small, medium, and large eosinophils with spherical and acidophilic granules; basophils with different concentrations of granules; small, medium, and large lymphocytes; monocytes; and azurophilic cells (Figs. 1, 2, and 3). The mature erythrocytes of T. merianae were shown to be morphologically similar to those of other reptiles (Canfield 1998): elliptical with a nucleus characterized by lumps of condensed chromatin and acidophilic cytoplasm. After Laür vital staining method, a large number of reticulocytes were observed with blue-colored precipitate in the cytoplasm indicating the presence of ribonucleoproteins. Basophils were characterized by metachromasy of the cytoplasmic granules after staining with toluidine blue. Azurophilic cells were distinguished from monocytes by the detection of abundant azurophilic cytoplasmic granules. These cells have also been described in other reptiles such as the turtle Gopherus agassizii (Alleman et al. 1992), the snake Elaphe obsoleta quadrivittata (Dotson et al. 1995), and the lizard Pogona vitticeps (Eliman 1997). Eosinophils
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were the rarest cells observed, as described for the snake Ophiophagus hannah (Salakij et al. 2002) and in other Squamata (Dotson et al. 1995). The cytoplasmic granules of azurophilic cells, as well as the granules of heterophils and eosinophils, show sudanophilia and a positive reaction to the myeloperoxidase. Glycogen, in the form of granules, was detected in the cytoplasm of thrombocytes, heterophils, and basophils, while proteins rich in basic polyaminoacids were found only in the granules of heterophils and eosinophils. Using electron microscopy, we could identify the following cells: thrombocytes, heterophils with granules of different sizes and electrondensities, eosinophils containing rounded granules bearing electrondense crystal-like material in several sizes, basophils with varied numbers of spherical and strongly electrondense granules, and azurophilic cells containing small electrondense granules. Similar results regarding the morphology and cytochemical aspects of the thrombocytes and leukocytes were also described by Oliveira et al. (1997) in alligators, by Alleman et al. (1992); Pellizzon et al. (2002); Knotková et al.(2002) in turtles, and Caxton-Martins and Nganwuchu (1978) and Martinez-Silvestre et al. (2005) in lizards. The lysosomal myeloperoxidase enzyme is known classically as a potent bactericidal agent related to the production of hypohaletos ions after reacting with hydrogen peroxide, which, in turn, is increased in the cellular defense due to the greater capacity for oxygen and the production of reactive oxygen species during phagocytic processes. This enzyme was detected in the azurophilic cells of the viperid snake Bothrops jararaca (Egami and Sasso 1988), in eosinophils and heterophils of the crocodile Caiman crocodilus yacare (Oliveira et al. 1998a,b), and also in Fig. 1 Tupinambis merianae. Photomicrographs of the main types of cells present in the peripheral blood. a Elliptical erythrocytes with a central, elliptical, and heterochromatic nucleus and acidophilic cytoplasm. Leishman; b reticulocytes with nuclei containing loose chromatin and acidophilic cytoplasm characterized by a net-shaped precipitate. Laür+Leishman; c elliptical thrombocyte, with a voluminous heterochromatic nucleus and hyalin cytoplasm with azurophilic granules (arrow); d a pair of heterophils showing bilobuled nuclei with heterogeneous chromatin and cytoplasm with spherical and rod-shaped granules, acidophilic and basophilic in chemical nature; e spherical eosinophil (center) with peripheral nucleus and cytoplasm with spherical acidophilic granules; f spherical basophil (center), with eccentric and heterochromatic nucleus and numerous spherical basophilic granules; g spherical basophil (center), with eccentric heterochromatic nucleus and scarce basophilic cytoplasmic granules; h spherical lymphocyte (center), with spherical nucleus, condensed chromatin, and basophilic cytoplasm with many projections; i monocyte (center) with peripheral nucleus with loose chromatin and basophilic cytoplasm with vacuoles; j azurophilic cell (center) with eccentric nucleus, chromatin condensed in lumps and cytoplasm with azurophilic compacted granules. Leishman; k basophil (center) with a large number of metachromatic granules; l basophil (center) with a small number of metachromatic granules. Toluidine blue. Bar=10 μm
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Comp Clin Pathol (2006) 15:169–174
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Fig. 2
Tupinambis merianae. Photomicrographs of the main types of cells present in the peripheral blood. a Heterophil (center) with cytoplasm rich in glycogen; b agglutinate thrombocytes (center) with glycogen-rich granules in the cytoplasm; c elliptical thrombocyte (arrow) showing compacted glycogen masses at the cell poles. Periodic acid-Schiff+Carazzi hematoxylin. d A pair of heterophils with granules strongly positive to sirius red. e Eosinophil (center)
Fig. 3 Tupinambis merianae. Transmission electron micrograph of the leukocyte buffy coat. a Thrombocytes showing elliptical nucleus (N) with internal folds (arrowheads) and cytoplasm with vesicles of the canalicular system (arrows); b heterophils with predominantly euchromatic nucleus (N) and cytoplasm with a large number of granules (g) with different shapes and electrondensities. The arrows point to the welldeveloped endoplasmic reticulum. Golgi apparatus (Go); c eosinophil with nucleus (N) showing peripheral heterochromatin and cytoplasm with different types of granules: large and roundish (arrowhead), small (arrow), and angled ones (asterisk). Some of the granules contain a strongly electrondense core in the shape of a small sphere or a small rod; d basophil with peripheral nucleus (N) with heterochromatin juxtaposed to the nuclear envelope and cytoplasm cointaining large (arrowhead) and small (arrows) spherical electrondense granules; e basophil with cytoplasm containing scarce spherical electrondense granules (arrowhead), mitochondria (arrow), endoplasmic reticulum (re), and free ribosomes. Nucleus (N); f azurophilic cell with nucleus (N) containing central euchromatin associated with the internal nuclear membrane and cytoplasm with small spherical granules (arrowhead), most of them very electrondense, free polyribosomes, and mitochondria. Bar=1 μm
173 strongly positive to sirius red. Sirius red+Carazzi hematoxylin. f A pair of heterophils positive to myeloperoxidase; g eosinophil (center) positive to myeloperoxidase; h azurophilic cell (center) positive to myeloperoxidase. O-toluidine-H2O2+Carazzi hematoxylin; i heterophil (center) with sudanophilic granules; j azurophilic cell (center) with granules weakly positive to sudan black B. Sudan black B+Carazzi hematoxylin. Bar=10 μm
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eosinophils of the turtle Phrynopys hilarri (Azevedo et al. 2002). The presence of glycogen and myeloperoxidase in the thrombocytes, heterophils, eosinophils, basophils, and azurophilic cells of T. merianae, as well as the formation of pseudopodes and phagocytic vacuoles detected both by light and electron microscopy, are compatible with the results of Azevedo and Lunardi (2003). These authors hypothesized that the recognition of existing chemical components in the medium by blood cells can trigger migration and phagocytic and bactericidal activity, all processes dependent on energy consumption. These cellular activities were confirmed by “in vitro” studies of blood cells in Alligator mississippiensis by Mateo et al. (1984) and in human blood cells by Zabucchi et al. (1990). The detection of basic proteins in T. merianae heterophils and eosinophils reveals the presence of cationic proteins possibly rich in arginine residues, as is classically known. These proteins are potent toxins for parasites, especially for helminthes, inactivating leukotrienes and causing the liberation of histamine by mast cells (Hamann et al. 1991). Lainson et al. (2003) demonstrated antihelminthic activity in eosinophils of the lizard Ameiva ameiva. Positive reaction for basic proteins was also described by Azevedo and Lunardi (2003) in the eosinophils of turtles. In this study, we have tried to gather the major morphological and cytochemical cellular features to characterize the blood cell types in the lizard T. merianae. Such characterization allows for the suggestion of specific functions for each one of these cell types, which can be confirmed in further studies using biochemical, immunological, and molecular biology methods.
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