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Caracterizacion morfo-citoquimica y ultraestructural de trombocitos del halcon buteo magnirostris (Gmelin, 1788) (Ave, falconiforme).

RESUMEN: La informacion hematologica basica sobre las aves en su habitat natural es limitado. Investigamos la sangre periferica de Buteo magnirostris, el numero de trombocitos y sus aspectos morfologicos, citoquimicos y ultraestructurales. Muestras de sangre fueron tratadas con los siguientes metodos: Leishman para analisis morfologico y metodos especificos para la caracterizacion de cuatro tipos de macromoleculas de diferente naturaleza quimica: Glicogeno, enzimas lisosomales (mieloperoxidasa y fosfatasa acida), poliaminoacidos basicos ricos en proteinas y fosfolipidos. El numero promedio de trombocitos fue 26.8 x [10.sup.3] [+ or -] 10,2 / mL. Estructuralmente los trombocitos presentaban predominantemente forma eliptica, con densa y agrupada cromatina nuclear. El citoplasma era claro, vacuolado y contenia uno o dos granulos azurofilicos morados en los polos. Los trombocitos presentaron reaccion citoquimica positiva para glucogeno, poliaminoacidos y fosfatasa acida. Ultraestructuralmente, los trombocitos se presentaban como celulas elipticas o esfericas conteniendo Golgi, con mucho reticulo endoplasmatico liso, pequenas mitocondrias, densos granulos osmiofilicos, microtubulos en la zona exterior de la celula y vacuolas grandes conectadas a traves del sistema canalicular o abiertas a la superficie.

PALABRAS CLAVE: 1. Aves; 2. Trombocitos; 3. Morfologia; 4. Citoquimica; 5. Ultrastructura.

Morpho-cytochemical and ultrastructural characterization of peripheral thrombocytes of roadside hawk Buteo magnirostris (Gmelin, 1788) (avian, falconiform)

SUMMARY: Basic hematological information about the avian in its natural habitat is limited. In this study we aimed at investigating, in the peripherical blood of the roadside hawk, Buteo magnirostris, the number of thrombocytes and their morphological, cytochemical and ultrastructural aspects. Blood smears were submited to the following methods: Leishman for morphological analysis and specific methods for the characterization of four kinds of macromolecules of different chemical nature: Glycogen, lysosomal enzymes (myeloperoxidase and the acid phosphatase), basic polyaminoacid rich proteins and phospholipids. The average number of thrombocytes was 26.8 x [10.sup.3] [+ or -]10,2 / [micro]L. Structurally the thrombocytes present predominantly elliptic shape, with dense and clumped nuclear chromatin. The cytoplasm is clear, vacuolated and may contain one or two purple azurophilic granules at the poles. The thrombocytes presented positive cytochemical reaction to glycogen, polyaminoacid and for acid phosphatase. Ultrastructurally, the thrombocytes appear as elliptical or spherical cells containing Golgi, much smooth endoplasmic reticulum, small mitochondria, dense osmiophilic granules, microtubules at the outermost zone of the cell and large vacuoles connected to the canalicular system or opened to the surface.

KEY WORDS: 1. Avian; 2. Thrombocyte; 3. Morphology; 4. Cytochemistry; 5. Ultrastructure.

INTRODUCTION

Avian thrombocytes appear as nucleated cells functionally similar to the human platelets. However, when morphologycal aspects are considered, they presented quite many differences.

In avian hematology, when performing the thrombocyte counting, as well as leukocyte differential counting, one must remember that it is easy to make mistakes considering the fact that small lymphocytes frequently resemble thrombocytes. Therefore, the thrombocytes quantification is extremely difficult. On the other hand, leukocytes and thrombocytes show morphological variation in different species.

Daimon & Uchida (1978), Ries et al., (1984), Swayne et al. (1986) purposed to study the morphological, structural, cytochemical and immunocytochemical aspects of thrombocytes in different avian species. In fact, it should be understood that the knowledge of many specific properties of thrombocytes allow us to establish correlation between structure and function.

In relation to the hematology of Brazilian specie of avian, the Buteo magnirostris, a roadside hawk, it was not possible to find any data in the specific literature. The first research about this subject concerns our previous study of blood granulocytes of this avian, whose manuscript has been sent for publication recently in the Journal of Submicroscopic Cytology and Pathology. In that article, we focused on some structural cytochemical and ultrastructural features of granulocytes. Now we decided to explore some parameters in relation to the peripherical thrombocytes. The total number of thrombocytes was also estimated.

Considering that progress in avian hematology is based largely on a few domesticated species (Bounous & Stedman, 2000) the results of this research could be of significative importance as comparative data of avian living in their natural habitat.

MATERIAL AND METHOD

14 adults specimens of avian, Buteo magnirostris (Gmelin, 1877), known popularly as the roadside hawk, belonging to the Falconform Order, were used in this study. The specimens were obtained from the Department of Parks and Green Reserved Areas of the Municipal Bureau of the Environment of Sao Paulo State, in Brazil.

For the performance of the structural, total counting of thrombocytes (TTC), cytochemical and ultrastructural analysis, 2.0ml of peripheral blood were collected from the brachial vein in the presence of EDTA 10%. Volumes of 0.5 ml were used for the following methods: Leishman for morphological description and for determination of total number of thrombocytes considering the number of thrombocytes in a thousand erithrocytes (Campbell, 1994), PAS according to Mc Manus (1946) for detection of glycogen; Sirius Red (Bogomoletz, 1980) for basic polyaminoacid detection; ortho-toluidine hydrogen peroxide (Jacobs, 1958) for myeloperoxidase identification; acid phosphatase demonstration according to Barka & Anderson (1962) and phospholipids using Sudan black B (Lison, 1960). The remaining volume was centrifuged (Anderson, 1965) and the buffy coat was promptly fixed in a mixture of glutaraldehyde and paraformaldehyde (Karnovsky, 1965) for studying by transmission electron microscope.

The thrombocytes number were determined according to Campbell, 1994 using the following simplified formula:

Estimated TTC = Number of Thrombocytes + Er = total of thrombocytes / [micro]l of blood/100

TCC: total counting of thrombocytes. Er: eritrhrocytes (The counting was carried out three times for each sample)

RESULTS

Morphological. The thrombocytes presented mostly eliptic aspect and a slightly spherical and oval shape as well. The basophilic nucleus occupies great part of its own volume in relation to the cytoplasma showing chromatine relatively condensed in a rough and grumous way. In eliptical thrombocytes, the cytoplasma presented itself hyaline of a vacuoloid aspect especially in the poles, sometimes showing in one of the poles one of two azurophilic granules clearly visible in most of the thrombocytes. (Fig. 1). Occasionally one can see thrombocytes tending to a spherical shape resembling limphocytes with vesicular nucleus roughly spherical sometimes showing basophilic cytoplasma (Fig. 2).

[FIGURES 1-2 OMITTED]

Determining the total Number of Thrombocytes/[micro]1. The total number of thrombocytes was determined through the indirect method in stained preparation using Leishman, recording the number of thrombocytes found as 1000 erithrocytes were counted perpassing all the smear. The counting was repeated three times for each sample. After a triple counting of the thrombocytes, the medium value and the standard deviation of the total number of thrombocytes per ml of blood (26.8 x [10.sup.3]/[micro]L [+ or -] 1.2.) were obtained.

Cytochemical. The cytochemical results are expressed in the Table I.

Ultrastructural. The thrombocytes when seen under the electron microscope presented predominatly eliptical, small with heterochomatic nucleus also eliptical and with an irregular surface.

One can see also big spherical thrombocytes with spherical nucleus and predominantly euchromatic. Both types presented cytoplasmatic characteristics alike, that is, presence of vacuoles of irregular format showing small electron dense granules located on their internal face. Some vacuoles presented structure like membrane in concentric lamellas resembling a figure of myelin. Prolife of mitochondria with electron dense matrix, complex of Golgi, endoplasmatic reticulum, marginal microtubules, a system of tubules, vesicles of the canalicular system, and an open system of canals connected to the surface were found (Fig. 3).

[FIGURE 3 OMITTED]

DISCUSSION

As in every hematological study, the influence of some factors, exogenous and endogenous, like the stress because of the capture and especially clinical conditions of the animal may interfere in the determining the hematimetrical indexes, including thrombocytes. Considering that all the samples of the peripherical blood of B. magnirostris were obtained in theirnatural habitat, that is, in the same conditions, the medium value found for the thrombocytes of this animal will be, certainly, as a parameter of reference for other species of fowls living in the same condition.

The overage of total number of thrombocytes made in triplicate for each sample revealed in this study, uniform values of 26.8 x [10.sup.3]/[micro]l such data are within the normal range of parameters also determined by Campbel, 1994, whose value reffered is 20.0-30,0 x [10.sup.3]/[micro]l for domestic fowls. Considering the uniform values obtained therefore, we believe we can admit this parameter can possibly correspond to that of a normal distribution.

According to the theory admitted by Bounous & Stedan, the thrombocytes of birds are cells morphologically distinct of platelets of the mammals, coming from mononuclear precursors in the lumen of medullary sinusoids, it's known that the principal organs of hematopoiese are made of in the beginning, along and after the incubation respectively: vitelline bag, spleen and bone marrow.

In Buteo magnirostris, this cell present itself predominantly eliptical, but ocasionally spherical or oval with slyghtly indented nucleus in one or both ends and in general following the shape of the cell. The morphological characterists frequently observed in the thrombocytes of this animal are: the presence of only one azurophil and voluminous granule clearly visible and vacuoles in one or both poles of this cell, like those observed by Janzarik (1981) and Bertram (1998) who characterized the thrombocytes morphologic and immuncytochemically. Besides this, it's possible to observe, in the peripherical blood of the animal in study, thrombocytes of irregular outline with spherical nucleus of light cromatin and basophilic cytoplasm, similar aspects to those observed by Janzarik & Morgenstern (1979) and Campbell (2000) where the authors mention the process of maturation of the thrombocytes relating them to the morphological changes in the blood stream. So, considering the similarities observed in relation to morphological aspects of the thrombocytes of this animal it is possible to admit that the final phases of maturation of this cell may occur in the circulation.

Among the thrombocytes found in the Buteo magnirostris especially, those small and spherical thrombocytes may be easily changed for small limphocytes of morphological aspect occasionally found in blood stream. Likewise, other authors like Janzarik & Morgenstern; Rossklopf & Woerpel (1983) also reported about similarities among small linphocytes and thrombocytes not only in fowls but also in reptiles, amphibians and fish (Moura, et al., 1998; Veiga et al., 2000).

Analysing the cytochemicals results, it was possible to observe, in the thrombocytes, the presence of glycogen in the one pole of the cytoplasma and posivity to basic poliaminoacids visible in only one voluminous cytoplasmatic granule and slightly positive reaction to acid phosphatase in a difuse way in the perinuclear region as observed by Swayne, Stockham & Johnson and by Egami & Sasso (1991) in fowls domestic.

Considering the energetic role of the glycogen (Cotran, 2000), the antibactericide activity of the cationic poliaminoacids (Evans et al., 1994) and the lisosomic function represented by the acid phosphatase (Andreasen & Latimer, 1990), it is possible to admit the inter relation among these substances and the phagocytic activity of the thrombocyte, once some authors like Chang & Hamilton (1979), Grecchi et al. (1980), Taffarel & Oliveira (1992), DaMatta et al. (1998) reported that these cells are found engaged not only in haemostatic process but also in phagocytic, endocytic and inflamatory processes.

As to the electronic microscopy, two distinct forms of thrombocytes were observed in peripherical blood of the Buteo magnirostris: elliptical and spherical, like those found by Maxwel & Trejo (1970) and Nirmalan et al. (1972). On the other hand, other researchers like Simpson (1967) and Sweeny & Carlson (1968) reported only one type of thrombocyte and normally in the ellipitcal forro. The elliptical form is, in general, smaller than the spherical one with a nucleus predominantly heterochromatic. The spherical thrombocytes show predominantly euchromatic nucleus. Both types present similar cytoplasmatic characteristics. The electronlucid vacuoles observed in the cytoplasm show inside them electron dense granules in the periphery or present several structures of membrane isolated or concentrically arranged as myelin figures. Occasionally vacuoles were also observed containing amorphous electron opaque material like those observed by Daimon et al. (1977), Daimon & Uchida (1978) and Pellizon (1996).

Taking into account that thrombocytes are cells related in a phagocitic or endocitic process (Taffarel & Oliveira; Damatta et al.), it is possible to admit that the vacuoles would be related to the process of digestion of the phagocitated material, following a degenaration as reported by Simpson (1967). Still observed and scattered along the cytoplasma of the thrombocytes in Buteo magnirostris were mytochondria with electron dense matrix, endoplasmatic reticulum, marginal bunches of microtubules, one system of tubules, vesicles derived from invagination of membrane and a system of open channels linked to the surface like those found by Maxwell & Trejo and Daimon & Uchida (1978).

Received: 10-08-2003

Accepted: 15-09-2003

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* Antenor Aguiar Santos; ** Adriana Marques Joppert da Silva; *** Vinicius Antonio Lima de Carvalho & **** Mizue Imoto Egami

* Master of Morphology UNIFESP-EPM, Brasil Professor of Morphology Department and Researcher of the Laboratory of Environmental Impact and Histophatology--Adventist University Center of Sao Paulo--UNASR Brazil

** Coordinator of Department of Parks and Green Reserved Areas of the Municipal Bureau of the Environment of S~o Paulo State, Brazil.

*** Master of Morphology UNIFESP-EPM, Sao Paulo, Brazil.

**** Associate Professor of Morphology Department--Histology Division and coordinator of Comparative Hematology Laboratory--UNIFESP-EPM

This research was financially supported by the Coordenacao de Aperfeicoamento de Pessoal de Ensino de nivel Superior (CAPES), Ministry of Education Science and Culture, Brazil.

Correspondence to:

Dr. Anterior Aguiar Santos

Estrada de I tapecerica, 3250

Bloco 03 Apto 11 - Judith Germania

05835-004 Sao Paulo-SP

BRAZIL

Tel: +55-11-58165630, Fax: +55-11-55764268

E-mail: antenoraguiar@hotmail.com
Table I. Cytochemical results of peripheral thrombocyte of
roadside hawk Buteo magnirostris.

Substances

              Glycogen (PAS)   Basic protein   Myeloperoxidase
Thrombocyte          +             +                -

              Acid phosphatase   Lipids
Thrombocyte       [+ or -]         -

Staining reactivity: - negative, [+ or -] faint, + positive
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Author:Aguiar Santos, Antenor; Marques Joppert da Silva, Adriana; Lima de Carvalho, Vinicius Antonio; Imoto
Publication:International Journal of Morphology
Date:Dec 1, 2003
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