Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration

Abstract

Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in vivo in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our in vivo imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our in vivo imaging led to the finding of a novel wound responsive mpeg1+ neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation.

Macrophages stimulate epicardial VEGFaa expression to trigger cardiomyocyte proliferation in larval zebrafish heart regeneration

“…We next sought to understand what role macrophages are playing in the regeneration of the larval heart, which occurs after just 48 hours 31, 32 . To test this, we used two completely different methods to induce macrophage-less hearts.…”

“…However, early revascularisation is critical for cardiomyocyte proliferation and is macrophage-dependent calling into question whether macrophages directly induce cardiomyocyte proliferaiton 17, 80 . Our larval model provided a unique opportunity to uncouple the angiogenic actions of macrophages from cardiomyocyte proliferation since larval hearts do not have supporting vasculature at this stage 32 . The increased proliferation we observed following injury demonstrates that larval hearts are truly regenerating and not recovering via normal developmental growth.…”

“…Heart images were acquired using 16X objective. The number of immune cells at the tail were quantified by counting from the caudal end of the vascular loop to the wound edge as performed by others 32, 101 . Tail images were acquired using 8X objective.…”

“…Larvae were subsequently digested in 1 μg/ml Proteinase K for 1 h. Larvae were re-fixed in 4% PFA for 20 mins and subsequently washed in PBT. TUNEL staining was performed using ApopTag Red In situ kit (MilliporeSigma) to label apoptotic cells, as described previously 32 . Stained hearts were imaged using LSFM.…”

Immunomodulation for optimal cardiac regeneration: insights from comparative analyses

“…3): Zebrafish neutrophils rapidly enter the site of injury but are more efficiently cleared in comparison to those in medaka 32 . An important mechanism, observed in the zebrafish using live imaging, is transmigration whereby neutrophils undergo reverse migration from the injured tissue back into the peripheral blood 82 . Prolonged inflammation leads to an increased CM loss, remodelling, fibrosis and rupture.…”