INTRODUCTION
Glucocorticoids are frequently used to treat inflammatory or immune disorders. However, a common but influential complication termed glucocorticoid-induced osteoporosis (GIOP) occurs in many patients receiving high-dose or long-term glucocorticoid therapy. Excessive glucocorticoid disrupts the balance between bone formation and bone resorption, leading to osteoporosis. Glucocorticoids have been demonstrated to promote bone resorption at the initial stage (in the 1st year of treatment) by increasing osteoclastogenesis and inhibiting the apoptosis of osteoclast precursor cells. In addition, glucocorticoids inhibit the differentiation and functions of osteoblasts and induce apoptosis of osteoblasts and osteocytes, leading to a decrease in bone formation[1]. During these processes, epigenetic modifications and mitochondrial bioenergetic alteration have been reported[2]. One research indicates that glucocorticoids induce the Lys-9 acetylation at the histone 3 and thus suppress Runt-related transcription factor 2 (Runx2) expression and osteoblast differentiation[3]. Besides, a prolonged glucocorticoid treatment usually triggers apoptosis of osteoblasts and osteocytes due to the disruption of mitochondrial bioenergetics that accumulates oxidative stress[45] or the induction of endoplasmic reticulum stress[67].
Zebrafish larvae are transparent and develop externally, allowing the progression of skeletal development and bone mineralization to be visualized after in vivo staining without invasive handling and animal sacrifice[8]. These advantages make zebrafish larvae suitable for studying skeletogenesis, building disease models, and screening anti-osteoporotic compounds. On the other hand, adult zebrafish's scales or fin rays are ideal for studying bone remodelling – the homeostasis maintained by osteoclast-mediated mineral resorption and osteoblast-mediated mineral deposition. Moreover, the quantity available and the live-imaging possibility highlight the ex vivo scale culture as an invaluable model for the high-throughput drug screen. The feasibility of tagging bone cells with fluorescent proteins through transgenesis makes zebrafish an ideal model for studying bone biology. In this review, we focus on the bone physiology and methodologies for building zebrafish as GIOP models.
ZEBRAFISH SHARE SIMILAR MOLECULAR TOOL KITS WITH MAMMALS IN SKELETAL DEVELOPMENT
Bone is a dynamic tissue that routinely undergoes remolding to maintain skeletal integrity[9]. The remolding process can be divided into five major phases: (1) the activation phase – mono-nucleated osteoclasts are recruited to the damaged bone surface; (2) the resorption phase – activated multi-nucleated osteoclasts start to remove the damaged bone; (3) the reversal phase – osteoclasts undergo apoptosis or leave the bone surface, and preosteoblasts are recruited to the digested bone surface; (4) the formation phase – mature osteoblasts lay down the nonmineralized organic matrix (osteoid), gradually embedding themselves into the osteoid, and undergo terminal differentiation into osteocyte; and (5) the mineralization phase – the osteoblasts and the osteocytes mineralize the osteoid, and the osteoblasts on the mineralized surface enter a quiescence state, which named as bone lining cells.
Although the entire bone remodeling processes have not been thoroughly documented in zebrafish, the essential cellular and molecular machinery are evolutionally conserved. The skeletal cells, such as bone-forming osteoblast and osteocyte, bone-resorbing osteoclast, and cartilage-forming chondroblast and chondrocyte, are all identified in zebrafish[10]. Signal pathways and genes involved in the differentiation of these skeletal cells also show similarities between zebrafish and mammals [Table 1]. For instance, Wingless-type MMTV integration site (Wnt) signaling is evolutionarily conserved in promoting osteoblastogenesis. Knockout of zebrafish wnt16, an ortholog of human WNT16, resulted in severe deformities and reduced bone mineral density[21]; similar phenotypes were also found in Wnt16−/− mice[22]. Although epigenetic control of zebrafish genes responding to glucocorticoids has not been explored, zebrafish genes involved in the differentiation and functions of osteoblast, osteocyte, or osteoclast are mostly conserved to their human counterparts [Tables 2 and 3]. These similarities make zebrafish a reasonable model for studying bone-related disorders.
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Table 1: Signaling pathways involved in zebrafish bone homeostasis
Table 2: Zebrafish-human orthologs implicated in osteoblast and osteocyte differentiation and function
Table 3: Zebrafish-human orthologs implicated in osteoclast differentiation and function
MODELING GLUCOCORTICOID-INDUCED OSTEOPOROSIS IN ZEBRAFISH LARVAE
The GIOP model using zebrafish larvae was first established in 2006 by Barrett et al. The authors demonstrated a significant mineral loss in the head bone after treating 25 μM prednisolone to the zebrafish larvae from 5 to 10 days post fertilization (dpf)[40]. Subsequent studies use either prednisolone (25 μM) or dexamethasone (10 or 15 μM) as the glucocorticoids to establish the GIOP models. The timing of glucocorticoid treatment in different studies could start from 3 to 5 dpf and stop at 9–10 dpf[4142]. To evaluate the therapeutic effect of candidate drugs against GIOP, colorimetric or fluorescent staining methods in conjunction with quantifying methods such as the staining area of head bones, integrated optical density of bones, or the numbers of calcified vertebrae could be adopted [Figure 1]A[404243]. Using the quantitative reverse-transcription polymerase chain reaction, the expression of genes related to the extracellular matrix, osteoblast, and osteoclast could be quantified in zebrafish larvae to investigate the molecular mechanism of anti-osteoporotic compounds[4244]. The possible impacts of prednisolone on zebrafish larvae have been demonstrated through upregulating the osteoclast-activity markers such as matrix metallopeptidase 9 (mmp9) and downregulating the preosteoblast and osteoblast markers such as sp7 transcription factor (sp7) and secreted phosphoprotein 1/osteopontin (spp1/opn), respectively[44]. Besides, prednisolone also suppresses the expression of various collagen genes, such as collagen, type XI, alpha 1a (col11a1a), resulting in cartilage defects[45], which is similar to that observed in osteoarthritis patients administrated with glucocorticoids[46].
Figure 1: Zebrafish models for glucocorticoid-induced osteoporosis. (A) The larval model for GIOP. After the glucocorticoid exposure, larvae could be stained for the examination of bone calcification. Inset on the right: calcein staining showing bone calcification. (B) The ex vivo scale-culture model for GIOP. The scales collected from adult zebrafish could be cultured in 96-well plates with glucocorticoids. The numbers and activity of osteoblast and osteoclast can be examined within 72 h post ex vivo culture. (C) The in vivo scale GIOP model. Scales are removed from the adult zebrafish using forceps. Then, the zebrafish will be exposed to the glucocorticoid to study the impact of glucocorticoids on scale regeneration. The numbers and activity of osteoblast and osteoclast could be examined. (D) Caudal fin amputation model for GIOP. Caudal-fin amputation is performed by surgical removal using a scalpel. The caudal fin amputated zebrafish is then exposed to glucocorticoids for weeks. The impact of glucocorticoids on bone regeneration could be addressed by examining the numbers and activity of osteoblast and osteoclast. (E) Fin-ray fracture model for GIOP. The fracture could be generated by using a needle or a pair of fin-tip forceps to crush the hemiray. After the fracture is generated, the zebrafish will be exposed to glucocorticoids to study the impact of glucocorticoids on fracture healing. (F) Skull trepanation model for GIOP. For the skull trepanation, a micro-drill is used to damage the os frontale of the calvarial bone. After the surgical intervention, the zebrafish will be exposed to glucocorticoids, and the bone healing process could be monitored. Abbreviations: F, os frontale; GC, glucocorticoid
MODELING GLUCOCORTICOID-INDUCED OSTEOPOROSIS IN ADULT ZEBRAFISH
Scales as a model for glucocorticoid-induced osteoporosis studies
The zebrafish skin is covered by a layer of calcified elasmoid scales, which belong to the dermal bone. The scales are stacked and attached by collagen fibrils comprising mainly two layers. The hyposquamal side (internal layer) is incompletely mineralized and composed of multiple layers of collagen fibrils. The episquamal side (external layer) is mineralized, containing a network of collagen fibrils[47]. Scleroblasts, the scale-forming osteoblasts derived from mesenchymal cells, are highly functionally similar to mammalian osteoblasts and express canonical osteoblastic markers[334849]. The scale also contains osteoclasts required to maintain the homeostasis of scale growth[39]. The zebrafish scale has a regenerative capacity and can completely regenerate within 21 days of removal[50].
The zebrafish scales are readily available and suitable for ex vivo and in vivo GIOP-related studies[485051]. Pasqualetti et al. established an ex vivo zebrafish scale culture model in 2012 for evaluating the osteoblast and osteoclast behavior [Figure 1]B[48]. They explanted scales from 6 months old zebrafish and placed them in a 96-well plate in Dulbecco's Modified Eagle Medium. They also observed apoptotic cells on the scale cultured for 72 h; accordingly, they concluded that assays for determining osteoblast and osteoclast activities, such as measuring alkaline phosphatase (Alp) and tartrate-resistant acid phosphatase (Trap) activities, should be performed within 72 h after explantation[48]. The scale culture could be considered an explanted organ and used to study the interaction among osteoblast, osteoclast, and the matrix, which may provide insights into bone regeneration and remodeling. This cost-efficient scale culture model has been used in a primary screen for anti-osteoporotic compounds[52]. The authors created a transgenic zebrafish expressing luciferase under the control of sp7, which is actively expressed in preosteoblast and osteoblast[52]. They explanted the scale from the transgenic fish and cultured them with selected compounds for 48 h, and then the corresponding luciferase activity was measured to quantify the changes in osteoblast differentiation[52]. This ex vivo zebrafish scale culture model preserves a natural environment for bone cells, which is more adequate to study the cell-cell interaction in a spacious manner compared to the traditional culture system.
As for the in vivo GIOP scale model, the adult zebrafish has to be exposed to 50 μM prednisolone for 15 days and the scales can be collected for further assays[51]. After the prednisolone exposure, the mineralization of scales is reduced, and the resorption of the scale lacunae is increased. The treatment of alendronate, an FDA-approved bisphosphonate anti-resorptive drug, alleviates the impact of prednisolone on the scale by decreasing the Trap activity and increasing the Alp activity[51]. Recent studies have adopted this in vivo scale GIOP model to evaluate the curative effect of the herbal mixture or compounds on the 9 months old zebrafish by treating them with 80 μM prednisolone for 14 days[5354].
The regenerating scales could also be used as an in vivo model to investigate the impact of glucocorticoids on bone mineralization [Figure 1]C[50]. In the regenerating scale GIOP model, the adult zebrafish are treated with prednisolone for 1 day prior to scale removal. After scale removal, the zebrafish are continually exposed to prednisolone, and the regenerated scales can be collected on the 8th or 21st days after prednisolone exposure[50]. The size and mineralization reduction, the activity of bone cells, and the matrix resorption of the scales from the prednisolone-treated zebrafish can be then measured[50]. This zebrafish scale model has been used to demonstrate the therapeutic effect of anti-osteoporotic compounds in treating GIOP[55]. A recent study showed that the intraperitoneal injection of dexamethasone to the adult zebrafish could shorten the administration time to less than 5 days, making this GIOP model more efficient and beneficial for a large-scale drug screening[56].
ESTABLISHMENT OF GLUCOCORTICOID-INDUCED OSTEOPOROSIS MODELS USING THE CAUDAL FIN
Zebrafish caudal fin is a nonmuscularized organ covered by the epidermis with strong regenerating capability and is supported by 16–18 principal bony rays. The fin ray is composed of bilateral parenthetical bones and is regularly divided into multiple bone segments[57]. A single layer of osteoblasts lines the inner and outer surfaces of the rays to secrete bone matrix[58]. In uninjured fins, osteoclasts may not be present in zebrafish caudal fins as the osteoclast-specific Trap activity is undetectable. However, Trap activity can first be detected 24 h after fin amputation, suggesting the recruitment of osteoclasts for the regeneration of fin rays[59].
The impact of glucocorticoids on caudal fin regeneration could be accessed by exposing the caudal-fin amputated adult zebrafish to 50 μM prednisolone for 4–6 weeks [Figure 1]D[6061]. Significant reductions in osteoblast proliferation and maturation and bone matrix mineralization were detected in prednisolone-treated zebrafish compared to control zebrafish after the caudal fin amputation, but osteoblast apoptosis was not observed. Prednisolone treatment reduces the number of osteoclasts and affects the migration of osteoclasts from the neighboring stump tissue to the wound site, thereby impairing bone resorption and then delaying bone regeneration[60]. Such an inhibitory effect of prednisolone against the osteoclast cells could be due to the high dosage of prednisolone treatment or the lack of the initial phase of enhancing osteoclastogenesis as reported in mammals. Continued prednisolone exposure could impair the expression of genes encoding extracellular matrix components and disrupt the machinery of macromolecule and vesicular transport[62]. A related study indicates that alendronate can restore the negative impact of glucocorticoids on caudal fin regeneration[63].
While GIOP increases the incidence of fractures[64], a bone crush model can also be established on the caudal fin rays to model fracture healing in patients with GIOP. The fracture can be easily generated on the hemiray segment using an injection needle or a pair of forceps [Figure 1]E[65]. Then, fractured rays undergo the healing process, including inflammation, chondrogenesis, ossification, and remodeling[66]. In the crush region, the elevated expression of spp1/opn could be recognized as an indicator for bone repairing[65]. It has also been found that excessive alendronate treatment impedes the process of bone healing and the removal of bone debris on the crush site, indicating the necessity of maintaining osteoclast activity for bone repair[67]. The fractured GIOP model could be readily established by treating the zebrafish with fractured fin-rays with 50 μM prednisolone for weeks[61]. On this basis, potential pro-healing compounds for GIOP-related fracture could be screened, and their mechanism of action could be analyzed.
CALVARIAE OF THE SKULL COULD BE USED FOR MODELING GLUCOCORTICOID-INDUCED OSTEOPOROSIS
The zebrafish calvariae, comprising the roof part of the skull, is suitable for studying bone healing under the condition of GIOP. The trepanation procedure could be used to create a wound by drilling a small hole in the os frontale [Figure 1]F. After the injury, the adult zebrafish are exposed to 50 μM prednisolone for 14 days. In the prednisolone-treated zebrafish, osteoblast number in the injured site is reduced, and the bone healing is delayed compared to the untreated control[60]. It is noteworthy that osteoblasts in the wound site undergo a distinct dedifferentiation process and become an essential source for bone healing[68]. This skull injury model could be helpful in studying the osteoblast dedifferentiation in zebrafish in the presence of glucocorticoids which can help in better understanding of bone healing mechanism under GIOP conditions.
IMAGING TECHNIQUES FOR EXAMINING BONE PHYSIOLOGY IN ZEBRAFISH
Techniques used to quantify bone mineralization have been extensively reviewed in zebrafish[6970]. Here, we summarize the tools applicable in zebrafish GIOP models. Whole-mount staining techniques are generally implemented to reveal the effect of glucocorticoids on bone mineralization and morphological changes in the larval or adult stages. Both alizarin red and calcein could be used to stain mineralized tissues in the head, axial skeleton, and fins in larval and adult stages. Alizarin red is mainly used as a chromogenic agent, whereas calcein stain is a fluorescent dye[8]. Both of which could be applied for live stain and observed by fluorescent microscopy[7172]. Cartilage could be stained by alcian blue, a polyvalent basic dye that could bind to the proteoglycan of cartilage at low pH[73]. Alcian blue staining is mainly applied in larval stages to assist the study of bone biology[74]. The dual stain of alizarin red and alcian blue could be performed to examine the overall morphology of both the hard bone and cartilaginous bones, respectively[75]. In adult zebrafish, making the sample transparent before performing cartilage and mineralization staining is critical. In addition to the whole-mount staining, transgenic fishes expressing fluorescent protein reporters of bone-related genes could be immensely helpful for speeding up the examination of bone cell differentiation by directly assessing under fluorescence microscopy[76]. For instance, transgenic zebrafish carrying a GFP reporter under the control of the sp7 promoter could be used to monitor the number of osteoblasts in vivo[607778].
Clinically, dual-energy X-ray absorptiometry is used to detect the bone mineral density of GIOP patients. Similarly, the X-ray skeletal images could be adopted as a primary tool to assess the deformity of fish bones[79]. For imaging the skeleton of zebrafish, a Faxitron MX-20 cabinet X-ray machine could be used[80]. Unfortunately, this technique is not suitable for larvae or juvenile zebrafish because of the resolution limitation[81]. To obtain much clearer and quantifiable images of zebrafish skeletons, micro-computed tomography (microCT) is a better option compared to X-ray photography. The 3-dimensional microCT imaging allows one to quantify the mineral density and bone morphology[82]. Besides, microCT imaging is demonstrated to be sensitive enough to quantify skeletal defects in the zebrafish model of skeletal dysplasia[83]. However, it is still difficult to detect the hypo mineralized bone of juvenile zebrafish under 30 dpf. For juvenile zebrafish older than 30 dpf, the bone structure could be seen by enhancing the detection sensitivity of microCT using a selective contrast agent, silver nitrate[83]. By contrast, transmission electron microscopy could also be applied to investigate the ultrastructure of bone matrix or cells at all stages[84]. Lastly, nano-indentation is used to assess the local mechanical properties of zebrafish vertebrae, including elastic modulus and hardness[85]. However, this technique is only suitable for extracted bone samples or biopsies.
CONCLUSIONS
To enhance the development of therapeutic drugs, it is essential to establish a fast, convenient, and reliable in vivo model. Current mammalian models used in GIOP research include mice, rats, rabbits, beagles, and ewes. One of the drawbacks of these mammalian models is that they are time-consuming to be established. For example, it takes 3 months to establish GIOP in rats through continuous oral administration[86] or subcutaneous injection of glucocorticoids[87]. As a result, it might take a long period of time to confirm the therapeutic effect of the candidate compounds. According to the genetic, physical, and physiological similarities between zebrafish and mammals in bone biology, zebrafish are suitable for modeling GIOP. The zebrafish models allow us to perform fast and efficient GIOP-related research with a relatively low experimental cost. The research result from zebrafish could be translated and applied to mammals well. For example, anti-GIOP compounds identified through zebrafish models[4277], such as tanshinol and salvianolic acid B, are also effective in mammalian models[8889]. Furthermore, using the zebrafish model for a preliminary drug screening could reduce the usage of mammalian models and comply with the three Rs – reduction, replacement, and refinement – ethical guidelines.
Financial support and sponsorship
This study was supported by the grants from Tzu Chi University (610400239-13) to M.D.L.
Conflicts of interest
There are no conflicts of interest.
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