Bulgarian Journal of Veterinary Medicine, 2021, 24, No 1, 2231 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, 2231.

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: , promegakaryocyte and (basophilic, acidophilic and thrombocytogenic). More intensive thrombopoiesis and morphologically differentiated cells were found in sternum samples. Key words: bone marrow, megakaryocyte, , thrombopoiesis, Wistar rat

INTRODUCTION

Bone marrow smears from rats usually is diminished as platelets production is contain a large number of 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 , 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 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 260280 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 45 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 number of basophilic Statistical analysis megakaryocytes in the femur, while the other cells were more represented in the Gathered data are represented as mean sternum. There was a significant diffe- values with standard deviation (mean ± 1 rence in the number of cells between the SD) and coefficient of variation (CV in sternum and the femur. %). Students t-test was used to analyse the statistical distribution. DISCUSSION

RESULTS Bone marrow analysis and collection is an integral part of a vast number of studies in Five different types of thrombopoietic the field of haematology and , cells were identified in the bone marrow predominantly from the femur (Soleimani of Wistar rats: megakaryoblast, promega- & Nadri, 2009). The haematopoietic karyocytes and megakaryocytes (basophi- system serves as a paradigm for under- lic, acidophilic and thrombocytogenic). standing much of adult stem cell biology Table 1 presents the most important (Challen et al., 2009). Therefore, much morphological characteristics of thrombo- effort is directed at developing the ex vivo poietic cells including cell shape as well expansion of functional megakaryocytes as cytoplasmic and nucleus colours. for exploratory and potential therapeutic Fig. 1 presents five identified and dif- purposes. Immunofluorescence analysis of ferentiated types of thrombopoietic cells megakaryocyte distribution shows that including and emperipolesis. these cells, although rare (less than 1%), All the cells are arranged in the "order" of are present in the bone marrow in the their maturation. Identification was niche of diaphysis, epiphysis and meta- performed by light microscopy. physis of the femoral sections, while a Fig. 2 shows megakaryocytes analysed slight increase is present within the by special microscopy using various types diaphysis of the femur (Malara et al., of light and wavelength. This analysis is 2014). used to detect "fine" cytoplasmic struc-

BJVM, 24, No 1 25 Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow

1 2

3 4

5 6 Fig. 1. Thrombopoietic lineage and osteoclast: 1. Megakaryoblast; 2. Promegakaryocyte; 3. Basofilic megakaryocyte; 4. Acidophilic megakaryocyte; 5. Thrombocytogenic megakaryocyte; 6. Emperipolesis with neutrophilic ;

26 BJVM, 24, No 1 D. Suljević, A. Hamzić, E. Islamagić, E. Fejzić & A. Alijagić

7.1 7.2 Fig. 1 (cont'd). Thrombopoietic lineage and osteoclast. 7.1 and 7.2: Osteoclast.

Fig. 2. Megakaryocyte under different light sources. Left: white light; middle: infrared (850 nm); right: near-ultraviolet (365 nm).

Table 2. Percentage of hematopoietic cells (mean ± SD) in sternum and femur of bone marrow.

Cells Megakaryoblast Promega- Basophilic Acidophilic Thrombocytogenic karyocyte megakaryocyte megakaryocyte megakaryocyte Femur 5.00±1.15 18.10±1.91 18.00±2.36 11.00±1.89 47.90±3.57 CV=23.09 CV=10.56 CV=13.09 CV=17.14 CV=7.46 Sternum 10.08±1.75 7.6*±1.51 10.09*±1.91 19.20*±3.05 51.50*±2.64 CV=16.21 CV=19.81 CV=17.54 CV=15.87 CV=5.12 CV – coefficient of variation; * P<0.05 vs femur. This study also encompassed megaka- ever, mature and larger megakaryocytes ryocyte analysis within the diaphysis of are always present near the sinusoids. This femur and sternum cavity. The maturity of tissue localisation is considered to be megakaryocyte and their size depend on appropriate for mature megakaryocytes, as their position in the vascular space. How- they elongate their cytoplasmic processes

BJVM, 24, No 1 27 Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow through endothelial fenestrae and release The size of the cell as well as the thrombocytes into circulation  vascular correlation between the cytoplasmic and space (Junt et al., 2007). Besides the bone nuclear ratio of hepatic megakaryocytes in marrow biopsy of the femur, for white rabbit from New Zealand show thrombopoietic cells, sternum bone certain differences as early as 22 days of marrow was also analysed. The analysed intrauterine life. This supports morpho- cell types have shown the significant functional mechanisms of hepatic mega- differences between femoral and sternal karyocytosis expressed on the fifteenth tissues. More were day of intrauterine life (Pacheco et al., identified in the sternum, suggesting 2002). increased haematopoietic intensity. The- Large liver megakaryocytes were refore, more mature megakaryocytes were observed during the twelfth day of foetal found such as the acidophilic and the development in mice, with significant thrombocytogenic types. According to the variations in size between twelfth and previous studies (Junt et al., 2007; Malara fifteenth days of pregnancy. During the et al., 2014), this could be due to the postnatal development, hepatic megaka- presence of a greater number of sinuses ryocytes show in their size, volume and and the proximity of megakaryocytes to N/C ratio several similarities with adult sinusoids in sternal bone marrow (Thon et animal bone marrow megakaryocytes al., 2010). (Pacheco et al., 2002). Active in different Circulating levels of thrombopoietin bones of rat bone marrow could be the (TPO) induce concentration-dependent result of a small number of mature proliferation and maturation of megaka- platelets, creating the largest megakaryo- ryocyte progenitors by binding to the cytes in the animal world. Five different c-Mpl receptor (Deutsch & Tomer, 2006). haematopoietic cells appear to take part in Mpl activity is regulated by a complex the formation of finite platelets as our cascade of signalling molecules that in- study shows. So far, there is no duce the action of specific transcription classification of these thrombopoietic factors to drive megakaryocyte prolifera- cells in this rat strain, but the identified tion and maturation (Liu et al., 2011; cells indicate the same similarities as in Potts et al., 2015). In situations where human thrombopoiesis (McCarthy, 2003). decrease in circulating numbers is In rats, several studies suggest megaka- evident, the resulting rise in TPO levels ryocytes presence but without specific allows a higher proportion of progenitors differentiation (Soleimani & Nadri, 2009; to commit to and/or complete megakaryo- Smailagić et al., 2013). By comparing the cyte development in bone marrow sizes of these cells to human counterparts, (Plutero & Kahr, 2016). Machlus et al. we observed larger rat cell dimensions. (2016) reported a potential positive feed- This certainly could be the result of a back mechanism whereby the platelet- different microenvironment of bone borne inflammatory cytokine chemokine marrow tissue, the lesser number of ligand 5 (CCL5) can stimulate megaka- sinusoids, the size of the animal and its ryocytes to produce platelets. life expectancy respectively (Kowata et Novel research demonstrated that IL- al., 2014). 21-mediated enhanced megakaryopoiesis mainly occurs in the bone marrow via IL-

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21R. IL-21 stimulatesf the proliferation of ture thrombocytes respectively (McCar- megakaryocyte progenitors via JAK3/ thy, 2003). When we observe cell STAT3 pathway (Benbarche et al., 2017). membrane shape in early development T cells mainly produce IL-21, indicating stages, we can recognise clear cell the regulation of megakaryopoiesis contours and the usual cell attributes. through adaptive immunity. Different cell shapes and structure are a It appears that IL-6 is a multifunc- product of their maturation process which tional interleukin and a strong promoter of conditions drastic changes in size and N/C megakaryocyte maturation process. This ratio. Dynamic pathway and sequence was observed (by our special microscopic maturation of hepatic megakaryocyto- methods) as increased size of mega- poiesis in rabbits, explains their different karyocytes as well as the increase in cell shapes. This process of full thrombocyte number. Additionally, ery- maturation takes three days to complete thropoietin along with IL contributes to (Pacheco et al., 2002). increased stimulation of processes in the Additionally, the nuclear membrane cytoplasm of megakaryocytes, leading to also possesses irregular structures. the final formation of thrombocytes (Lane Intranuclear compartments observed in et al., 2000; Thon & Italiano, 2010). In this study were usually with oval and summary, taking above-mentioned studies reniform shape with irregular lobular into account, these large thrombopoietic structure. The shape of these components cells along with cell, cytoplasm and nuclei clearly deviates from the standard circular size and ratio, are a consequence of early shape. Indeed, these irregular nuclear factor activation. These factors include structures and contours are a unique trait erythropoietin, thrombopoietin and IL-6 of acidophilic and thrombocytogenic during intrauterine development. This megakaryocytes (Centurione et al., 2004). early activation of factors mentioned In conclusion, we identified five types above results in very large rat mega- of haematopoietic thrombocyte precursors karyocytes formation at a very early stage in rat bone marrow. In contrast to femoral of rat hematopoiesis. This also shows that diaphysis, increased haematopoietic cell factors present in intrauterine haema- production and increased mature megaka- topoiesis could be differently expressed in ryocytes number were observed in sternal adult bone marrow cells, along with IL-6 . bone marrow samples. Thrombocyte pre- Subcutaneous application of IL-6 (10 cursor maturation process encompasses an µg/day) causes cytoplasmic microtubule increase in overall cell dimensions, produ- bundle formations which proportionally cing a very large terminal mature cell, so lead towards thrombocyte formation in our research, megakaryocytes represent (Kaser et al., 2001). This suggests IL-6 as the largest haematopoietic cells in Wistar a megakaryocyte forming and thrombo- rat haematopoiesis. A smaller number of cytogenic megakaryocyte fragmenting thrombocyte precursors and the terminal factor which increases mature thrombo- large mature cell could be the result of cytes number. As for thrombopoietin, it several factors such as number and the appears to be responsible for complete close proximity of bone marrow sinusoids, megakaryocyte maturation through and life longevity of rats as one of the granule formation, membrane demarcation critical factors for intensified haemato- and cytoplasmic fragmentation into ma- poiesis.

BJVM, 24, No 1 29 Haematopoietic thrombocyte precursors in rat femoral and sternal bone marrow

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ential analysis of rat bone marrow by flow cytometry. Comparative Haematology In- ternational, 10, 97. Paper received 25.04.2019; accepted for Smajilagić, A., M. Aljičević, A. Redžić, S. publication 01.09.2019 Filipović & A. Lagumdžija, 2013. Rat bone marrow stem cells isolation and cul- ture as a bone formative experimental sys- tem. Bosnian Journal of Basic Medical Sciences, 13, 2730. Correspondence: Soleimani, M. & S. Nadri, 2009. A protocol for isolation and culture of mesenchymal Damir Suljević, PhD, Associate Professor stem cells from mouse bone marrow. Department of Biology, Faculty of Science, Nature Protocols, 4, 102–106. University of Sarajevo, Sarajevo, Bosnia and Herzegovina Suljević, D., F. Filipić, & E. Islamagić, 2018. Tel.: 0038733723776 Emperipolesis: Sternal and femoral micro- e-mail: [email protected] environment induces megakaryiocyte

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