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Original Article HAEMATOPOIETIC THROMBOCYTE PRECURSORS Bulgarian Journal of Veterinary Medicine, 2021, 24, No 1, 2231 ISSN 1311-1477; DOI: 10.15547/bjvm.2019-0063 Original article HAEMATOPOIETIC THROMBOCYTE PRECURSORS IN RAT FEMORAL AND STERNAL BONE MARROW D. SULJEVIĆ1, A. HAMZIĆ1, E. ISLAMAGIĆ1, E. FEJZIĆ2 & A. ALIJAGIĆ1 1Department of Biology, Faculty of Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina; 2Institute for Transfusion Medicine of FBiH, Sarajevo, Bosnia and Herzegovina Summary Suljević, D., A. Hamzić, E. Islamagić, E. Fejzić & A. Alijagić, 2021. Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow. Bulg. J. Vet. Med., 24, No 1, 2231. This research presents the first findings on thrombopoiesis for Wistar rats. Haemopoietic cells from the femur and the sternum were analysed by light microscopy in combination with infrared and near- ultraviolet light for fine cytoplasmic structure analysis. Five main types of thrombocyte precursor cells were identified in the bone marrow samples: megakaryoblast, promegakaryocyte and megakaryocyte (basophilic, acidophilic and thrombocytogenic). More intensive thrombopoiesis and morphologically differentiated cells were found in sternum samples. Key words: bone marrow, megakaryocyte, platelets, thrombopoiesis, Wistar rat INTRODUCTION Bone marrow smears from rats usually is diminished as platelets production is contain a large number of megakaryocytes shifted from the spleen to some other accounting for 0.40–0.77% of total organ, like bone marrow (Bolliger, 2004). nucleated cells in rat bone marrow (Saad Large megakaryocytes could be a et al., 2000). Density and distribution of product of an early thrombopoietic phase megakaryocytes vary with technique and occurring in the first days of pregnancy as among smears; however, there are no observed in the mammalian order of published results regarding the precise rodents (Rodentia) (Pacheco et al., 2002). number of megakaryocytes in rats. During The increase in the size of hepatic late foetal and early postnatal life, the megakaryocytes might be a result of renal, spleen is active in megakaryocytopoiesis. hepatic and immunological development These cells are more abundant in the during foetal stages. This situation may immature animal (neonates and juveniles), also be the cause of a more increased and but were never found in the white pulp of favoured production of humoral stimu- spleen (Raval et al., 2014). This function lation factors towards large cell oriented D. Suljević, A. Hamzić, E. Islamagić, E. Fejzić & A. Alijagić megakaryocytopoiesis. In adult stages, attributed simply to a random movement proximity to sinusoids and microenviron- and adherence of cells to the megaka- ment of the bone marrow itself have a ryocytes in mice (Centurione et al., 2004). greater role in megakaryocytopoiesis and Megakaryocytes form platelets which cell differentiation in general (Pacheco et play fundamental role in haemostasis and al., 2002). coagulation processes. Haematopoietic As in other species, rat megaka- tissues, depending on the physiological ryocytes are the largest haematopoietic anatomic characteristics, have a different cells. Mature megakaryocytes in rats are rate of haematopoietic cell production. multinucleated, with nuclei often fused The aim of this study was to identify and into a lobulated mass. Cytoplasm is to quantify haematopoietic precursors of abundant, light blue, and filled with fine megakaryocytes based on their morpho- eosinophilic granules (Bolliger, 2004). logy in the bone marrow of sternal and Younger megakaryocytes are smaller, femoral bone in Wistar rats. Also, the rate with higher nuclear:cytoplasmic (N:C) of megakaryocytes production based on ratios, more basophilic cytoplasm, and the number of less or more mature forms fewer nuclei. Maturation should be of megakaryocytic precursors was orderly, with a lower number of immature compared. than mature forms. Megakaryocytes should not be confused with osteoclasts, MATERIALS AND METHODS which have separated nuclei and less abundant cytoplasm (Bolliger, 2004). This study was conducted at the The ultrastructural observations sho- Laboratory for Physiology, Faculty of wed that megakaryocytes in the bone Natural Sciences and Mathematics, marrow of rats are characterised by the University of Sarajevo, Bosnia and Herze- absence of destruction of engulfed cells govina. All animals were well cared for and phagosome formation in the according to the Animal Protection and megakaryocytes, which was defined as Welfare Law of Bosnia and Herzegovina emperipolesis (Suljević et al., 2018). (“Službene Novine” 25/09). Morphologically, occasional megakaryo- cytic emperipolesis can be detected in the Animals and breeding normal bone marrow. Emperipolesis was Our main sample consisted of ten observed only in mature stage III laboratory-bred adult Wistar rats (Rattus megakaryocytes. The incidence of mega- norvegicus s. Wistar, n=10), six males and karyocyte emperipolesis was markedly four females. Sample animals were kept in increased in ageing rats that showed a vivarium (individual medium plexiglass haematopoietic cell hyperplasia in the cages at 25 °C with 12/12 hours of light bone marrow (Bolliger, 2004). There is no and dark cycle) and were fed pelleted evidence of cellular damage to the food (Oxbow Essentials) with water ad engulfed marrow cells or megakaryocytes. libitum. At the time of biopsy, animals Possible explanation is that the were approximately between 10 and 11 megakaryocyte cytoplasm might provide a weeks old and at the same developmental „secure place“ for normal granulocytes stage. The weighing procedure was under an unfavourable bone marrow necessary for the anaesthetic dosage and environment or that emperipolesis is euthanasia. The average body mass of rats BJVM, 24, No 1 23 Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow was 283.70±13.87 g. The largest number were used. Rectangular shaped cut of individuals had body mass 260280 g, (comprising of four precise cuts) was and three individuals had an average made on bone samples. Cut regions on weight of about 300 g. femur were from most of the femoral body (shaft) area while sternum was cut from Bone marrow extrapolation and staining manubrium to xiphisternal joint. Access to Selected animals were anaesthetised first bone marrow was achieved by lifting the by sufentanil/medetominide (subcutane- rectangular cut-out of the bone. Bone ously 50/150 µg/kg) and euthanised by marrow was removed physically by point doubling the dosage to comply with gauge needle (21G/0.8×40 mm; Semikem) proper ethical procedures. Incisions by and placed on the microscopic slide. scalpel were made in the thoracic regions Using a small glass rod, the marrow of the chest and hind legs to remove sample was rolled over the slide in a slow sternum and femurs. Tissue components paced and careful linear movement (touch were removed, and bones were thoroughly technique). After the samples had dried at cleaned. Incision spots were marked room temperature, they were stained by before the cutting. To open the bone May-Grünwald-Giemsa stain (Semikem) segments and avoid excessive damage to and Leders stain (Semikem) for evaluation the bone marrow, surgical grade scalpels of peroxidase activity. Table 1. Morphological characteristics of cells and nucleus of respective cell types Shape Staining property Cell Nucleus Cytoplasm Nucleus Megakaryoblast Oval with irregular outlines, Large nucleus with Basophilic Basophilic the smallest cell in irregular structure, megacaryocytic cell line occupies 95% of cell Promegakaryocyte Oval with irregular outlines, Consists of 45 nuclear Basophilic Basophilic larger than nucleus in segments megakaryoblast Basophilic megakaryocyte Irregular round shape, larger Several nuclear Basophilic, light Light blue than promegakaryocyte segments in group blue at the cell borders Acidophilic megakaryocyte Irregular elongated shape, Large amount of Light blue around Acidophilic, larger than basophilic separated nucleus nucleus and light non-homoge- megakaryocyte segments, not grouped red in other parts nated Thrombocytogenic megakaryocyte The largest cell in in Fragmented and integral Bright acidophilic Very bright megacaryocytic cell line, nuclei scattered in the acidophilic irregular „amoeboid“ shape cytoplasm 24 BJVM, 24, No 1 D. Suljević, A. Hamzić, E. Islamagić, E. Fejzić & A. Alijagić Microscopic analysis ture. The analysis showed that there were no specific characteristics in the cyto- Main cell identification and analysis were plasm that would require additional cell performed on a light microscope (Olym- identification methods. This suggests that pus BX41) equipped with a digital camera light microscopy is sufficient for the (Olympus DP12). Image processing was identification of megakaryocytes, but done in a licensed software (Olympus special microscopy is an excellent tool for DP12 Soft DP12-CB Ver.01.01.01.42.® visualising cytoplasmic maturation and Olympus Corp.). From each sternum and fragmentation. femur sample, 100 cells were included for Table 2 shows the percentage of hae- identification and sequestial morpholo- matopoietic cells in the bone marrow of gical analysis. Additional sample analysis Wistar rats. Identification was analysed in was performed on a digital microscope the sternum and femur. The most (Bresser LCD Digital) with three light numerous cells in both tissues were sources (white light, infrared at 850 nm thrombocytogenic megakaryocytes. There and near ultraviolet at 365 nm). was a greater
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