Matrix metalloproteinases (MMPs) are key enzymes in the turnover of extracellular matrix in health, disease, development and regeneration. We have studied zebrafish scale regeneration to ascertain the role of MMP-2 and MMP-9 in these processes. Scales were plucked from the surface of anaesthetised adult male zebrafish, and the scales that regenerated in the scale pocket were recovered at various time points after plucking. Analyses consisted of (i) mmp-9 in situ hybridisation; (ii) MMP-9+TRAcP double-staining; (iii) qRT-PCR for mmp-2 and mmp-9; (iv) zymography for gelatinolytic activity and (v) a hydroxyproline assay. We found that mmp-9 positive cells were confined to the episquamal side of the scales. Ontogenetic scales had irregular clusters of mono- and multinucleated mmp-9 expressing cells along their lateral margins and radii. During regeneration, mmp-9 positive cells were seen on the scale plate, but not along the lateral margins. Double staining for TRAcP and MMP-9 revealed the osteoclastic nature of these cells. During early scale regeneration, mmp-2 and mmp-9 transcripts increased in abundance in the scale, enzymatic MMP activity increased and collagen degradation was detected by means of hydroxyproline measurements. Near the end of regeneration, all of these parameters returned to the basal values seen in ontogenetic scales. These findings suggest that MMPs play an important role in remodelling of the scale plate during regeneration, and that this function resides in mononucleated and multinucleated osteoclasts which co-express TRAcP and mmp-9. Our findings suggest that the fish scale regeneration model may be a useful system in which to study the cells and mechanisms responsible for regeneration, development and skeletal remodelling.
Silica nanoparticles can be efficiently employed as carriers for therapeutic drugs in vitro. Here, we use zebrafish embryos as a model organism to see whether mesoporous silica nanoparticles (MSNPs) can be incorporated to deliver compounds in vivo. We injected 35-40 nL (10 mg/mL) of custom-synthesized, fluorescently-tagged 200 nm MSNPs into the left flank, behind the yolk sac extension, of 2-day-old zebrafish embryos. We tracked the distribution and translocation of the MSNPs using confocal laser scanning microscopy. Some of the particles remained localized at the injection site, whereas others entered the bloodstream and were carried around the body. Embryo development and survival were not significantly affected by MSNP injection. Acridine orange staining revealed that MSNP injections did not induce significant cell death. We also studied cellular immune responses by means of lysC::DsRED2 transgenic embryos. MSNP-injected embryos showed infiltration of the injection site with neutrophils, similar to controls injected with buffer only. In the same embryos, counterstaining with L-plastin antibody for leukocytes revealed the same amount of cellular infiltration of the injection site in embryos injected with MSNPs or with buffer only. Next, we used MSNPs to deliver two recombinant cytokines (macrophage colony-stimulating factor and receptor for necrosis factor ligand) to zebrafish embryos. These proteins are known to activate cells involved in bone remodeling, and this can be detected with the marker tartrate-resistant acid phosphatase. Coinjection of these proteins loaded onto MSNPs produced a significant increase in the number of tartrate-resistant acid phosphatase-positive cells after 2-3 days of injection. Our results show that MSNPs can be used to deliver bioactive compounds into zebrafish larvae without producing higher mortality or gross evidence of teratogenicity.
This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX-induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0-5 hours) of wound-healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up-regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life.
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