Skeletal muscle regeneration after extensive cryoinjury of caudal myomeres in adult zebrafish.

Skeletal muscles can regenerate after minor injuries, but severe structural damage often leads to fibrosis in mammals. Whether adult zebrafish possess the capacity to reproduce profoundly destroyed musculature remains unknown. Here, a new cryoinjury model revealed that several myomeres efficiently regenerated within one month after wounding the zebrafish caudal peduncle. Wound clearance involved accumulation of the selective autophagy receptor p62, an immune response and Collagen XII deposition. New muscle formation was associated with proliferation of Pax7 expressing muscle stem cells, which gave rise to MyoD1 positive myogenic precursors, followed by myofiber differentiation. Monitoring of slow and fast muscles revealed their coordinated replacement in the superficial and profound compartments of the myomere. However, the final boundary between the muscular components was imperfectly recapitulated, allowing myofibers of different identities to intermingle. The replacement of connective with sarcomeric tissues required TOR signaling, as rapamycin treatment impaired new muscle formation, leading to persistent fibrosis. The model of zebrafish myomere restoration may provide new medical perspectives for treatment of traumatic injuries.

Autologous Blood Transfusion During Elective Surgery in a Candidate for Living Donor Transplantation: A Case Report.

BACKGROUND: Blood transfusions are risk factors for alloimmunization and unfavorable outcomes in solid organ transplant recipients. PURPOSE: We propose the adoption of autologous blood transfusion (ABT) in transplant candidates and recipients referred to elective surgery. METHODS: We present a case of a 45-year-old man with chronic kidney disease stage 5 due to polycystic kidney disease, who was qualified for a native kidney nephrectomy (NKN) before kidney transplantation. Before the scheduled surgery, the patient was referred to a blood donation center for blood collection. RESULTS: During 2 consecutive visits, autologous blood was collected uneventfully, and this allowed for the preparation of 2 units of red blood cell concentrates and a unit of plasma. Pre- and post-donation hemoglobin values were 11.9 and 10.4 g/dL, respectively. The NKN procedure was complicated by intra-abdominal bleeding from an accessory aberrant artery of the kidney. Hemoglobin dropped to 6.8 g/dL and was treated with ABT, followed by artery embolization. This allowed for an increase of hemoglobin to 8.3 mg/dL and avoidance of allotransfusion. Six weeks after NKN, the patient underwent successful kidney transplantation from a living donor. Panel reactive antibodies before transplantation were 0%, and graft function has been excellent during 20 months of observation. CONCLUSION: An autologous blood collection is a feasible option for patients with chronic kidney disease. ABT should be considered the procedure of choice when qualifying potential waiting list candidates and solid organ recipients for elective surgeries.

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish.

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Regeneration of the dermal skeleton and wound epidermis formation depend on BMP signaling in the caudal fin of platyfish.

Fin regeneration has been extensively studied in zebrafish, a genetic model organism. Little is known about regulators of this process in distant fish taxa, such as the Poeciliidae family, represented by the platyfish. Here, we used this species to investigate the plasticity of ray branching morphogenesis following either straight amputation or excision of ray triplets. This approach revealed that ray branching can be conditionally shifted to a more distal position, suggesting non-autonomous regulation of bone patterning. To gain molecular insights into regeneration of fin-specific dermal skeleton elements, actinotrichia and lepidotrichia, we localized expression of the actinodin genes and bmp2 in the regenerative outgrowth. Blocking of the BMP type-I receptor suppressed phospho-Smad1/5 immunoreactivity, and impaired fin regeneration after blastema formation. The resulting phenotype was characterized by the absence of bone and actinotrichia restoration. In addition, the wound epidermis displayed extensive thickening. This malformation was associated with expanded Tp63 expression from the basal epithelium towards more superficial layers, suggesting abnormal tissue differentiation. Our data add to the increasing evidence for the integrative role of BMP signaling in epidermal and skeletal tissue formation during fin regeneration. This expands our understanding of common mechanisms guiding appendage restoration in diverse clades of teleosts.

Platyfish bypass the constraint of the caudal fin ventral identity in teleosts.

BACKGROUND: The caudal fin of teleosts is characterized by dorsoventral symmetry. Despite this external morphology, the principal rays of this appendage connect to bones below the notochord, indicating the ventral (hypochordal) identity of this organ. RESULTS: Here, we report that this typical architecture of the caudal fin is not fully conserved in the platyfish (Xiphophorus maculatus) and the guppy (Poecilia reticulata), representatives of the Poeciliidae family. We show that in these species, 3-4 principal rays connect to bones above the notochord, suggesting an epichordal contribution. Consistently, as examined in platyfish, dorsal identity genes zic1/4 were highly expressed in these rays, providing molecular evidence of their epichordal origin. Developmental analysis revealed that the earliest rays above the notochord emerge at the 10-ray stage of fin morphogenesis. In contrast to zebrafish and medaka, platyfish and guppies display a mirrored shape of dorsal and ventral processes of the caudal endoskeleton. Our study suggests that an ancestral bauplan expanded in poeciliids by advancing its symmetrical pattern. CONCLUSION: The platyfish evolved a fin architecture with the epichordal origin of its upper principal rays and a high level of symmetry in the caudal endoskeleton. This innovative architecture highlights the adaptation of the teleost skeleton.

Hydrodynamic stress and phenotypic plasticity of the zebrafish regenerating fin.

Understanding how extrinsic factors modulate genetically encoded information to produce a specific phenotype is of prime scientific interest. In particular, the feedback mechanism between abiotic forces and locomotory organs during morphogenesis to achieve efficient movement is a highly relevant example of such modulation. The study of this developmental process can provide unique insights on the transduction of cues at the interface between physics and biology. Here, we take advantage of the natural ability of adult zebrafish to regenerate their amputated fins to assess its morphogenic plasticity upon external modulations. Using a variety of surgical and chemical treatments, we could induce phenotypic responses to the structure of the fin. Through the ablation of specific rays in regenerating caudal fins, we generated artificially narrowed appendages in which the fin cleft depth and the positioning of rays bifurcations were perturbed compared with normal regenerates. To dissect the role of mechanotransduction in this process, we investigated the patterns of hydrodynamic forces acting on the surface of a zebrafish fin during regeneration by using particle tracking velocimetry on a range of biomimetic hydrofoils. This experimental approach enabled us to quantitatively compare hydrodynamic stress distributions over flapping fins of varying sizes and shapes. As a result, viscous shear stress acting on the distal margin of regenerating fins and the resulting internal tension are proposed as suitable signals for guiding the regulation of ray growth dynamics and branching pattern. Our findings suggest that mechanical forces are involved in the fine-tuning of the locomotory organ during fin morphogenesis.

Persistent Ventricle Partitioning in the Adult Zebrafish Heart.

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF), both emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled, loxP reporter results in the predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

Multiple cryoinjuries modulate the efficiency of zebrafish heart regeneration.

Zebrafish can regenerate their damaged hearts throughout their lifespan. It is, however, unknown, whether regeneration remains effective when challenged with successive cycles of cardiac damage in the same animals. Here, we assessed ventricular restoration after two, three and six cryoinjuries interspaced by recovery periods. Using transgenic cell-lineage tracing analysis, we demonstrated that the second cryoinjury damages the regenerated area from the preceding injury, validating the experimental approach. We identified that after multiple cryoinjuries, all hearts regrow a thickened myocardium, similarly to hearts after one cryoinjury. However, the efficiency of scar resorption decreased with the number of repeated cryoinjuries. After six cryoinjuries, all examined hearts failed to completely resolve the fibrotic tissue, demonstrating reduced myocardial restoration. This phenotype was associated with enhanced recruitment of neutrophils and decreased cardiomyocyte proliferation and dedifferentiation at the early regenerative phase. Furthermore, we found that each repeated cryoinjury increased the accumulation of collagen at the injury site. Our analysis demonstrates that the cardiac regenerative program can be successfully activated many times, despite a persisting scar in the wounded area. This finding provides a new perspective for regenerative therapies, aiming in stimulation of organ regeneration in the presence of fibrotic tissue in mammalian models and humans.

Lymphatic vessels help mend broken hearts.

Experiments on zebrafish show that the regeneration of the heart after an injury is supported by lymphatic vessels.

Distribution and Restoration of Serotonin-Immunoreactive Paraneuronal Cells During Caudal Fin Regeneration in Zebrafish.

Aquatic vertebrates possess diverse types of sensory cells in their skin to detect stimuli in the water. In the adult zebrafish, a common model organism, the presence of such cells in fins has only rarely been studied. Here, we identified scattered serotonin (5-HT)-positive cells in the epidermis of the caudal fin. These cells were distinct from keratinocytes as revealed by their low immunoreactivity for cytokeratin and desmosome markers. Instead, they were detected by Calretinin (Calbindin-2) and Synaptic vesicle glycoprotein 2 (SV2) antibodies, indicating a calcium-regulated neurosecretory activity. Consistently, electron microscopy revealed abundant secretory organelles in desmosome-negative cells in the fin epidermis. Based on the markers, 5-HT, Calretinin and SV2, we referred to these cells as HCS-cells. We found that HCS-cells were spread throughout the entire caudal fin at an average density of 140 cells per mm2 on each fin surface. These cells were strongly enriched at ray bifurcations in wild type fins, as well as in elongated fins of another longfin mutant fish. To determine whether hydrodynamics play a role in the distribution of HCS-cells, we used an interdisciplinary approach and performed kinematic analysis. Measurements of particle velocity with a fin model revealed differences in fluid velocities between bifurcated rods and adjacent non-bifurcated regions. Therefore the accumulation of HCS-cells near bone bifurcations may be a biological adaptation for sensing of water parameters. The significance of this HCS-cell pattern is reinforced by the fact, that it is reestablished in the regenerated fin after amputation. Regeneration of HCS-cells was not impaired by the chemical inhibition of serotonin synthesis, suggesting that this neurotransmitter is not essential for the restorative process. In conclusion, our study identified a specific population of solitary paraneurons in the zebrafish fin, whose distribution correlates with fluid dynamics.

Intrathoracic Injection for the Study of Adult Zebrafish Heart.

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

Zebrafish fin regeneration involves transient serotonin synthesis.

The zebrafish is a vertebrate organism capable of regenerating many of its organs. Notably, it can undergo epimorphic regeneration of its fins after amputation. This process occurs through the formation of a wound epithelium and the dedifferentiation of mesenchymal and bone-forming cells, which form a proliferative blastema. Here, we report that the entry into the regenerative process involves the local synthesis of serotonin (5-hydroxytryptamine, 5-HT) in the injury-associated tissue. One day after wounding, intracellular accumulation of serotonin was induced in the stump below the amputation plane. During blastema formation, serotonin was detected in the mesenchyme at the vicinity of the amputation plane and in the apical wound epithelium. During the advanced outgrowth phase, this monoamine was no longer present in the blastema, suggesting a temporal involvement of serotonin in the postinjury area. We show the expression of two serotonin synthesizing enzymes, tryptophan hydroxylase 1a and 1b in the blastema, suggesting the local production of this monoamine. Neither depletion of serotonin by chemical inhibition of tryptophan hydroxylase, nor ectopic administration of this monoamine affected fin regeneration, indicating it does not play a role during this process. Finally, we found that the presence of serotonin during regeneration depends on fibroblast growth factor and retinoic acid signaling. Overall, our study demonstrates that the initiation of fin regeneration is associated with a transient synthesis of serotonin in the regrowing tissue.

Ciliary neurotrophic factor stimulates cardioprotection and the proliferative activity in the adult zebrafish heart.

Unlike mammals, adult zebrafish can regenerate their hearts after injury via proliferation of cardiomyocytes. The cell-cycle entry of zebrafish cardiac cells can also be stimulated through preconditioning by thoracotomy, a chest incision without myocardial damage. To identify effector genes of heart preconditioning, we performed transcriptome analysis of ventricles from thoracotomized zebrafish. This intervention led to enrichment of cardioprotective factors, epithelial-to-mesenchymal transition genes, matrix proteins and components of LIFR/gp130 signaling. We identified that inhibition of the downstream signal transducer of the LIFR/gp130 pathway through treatment with Ruxolitinib, a specific JAK1/2 antagonist, suppressed the cellular effects of preconditioning. Activation of LIFR/gp130 signaling by a single injection of the ligand Cilliary Neurotrophic Factor, CNTF, was sufficient to trigger cardiomyocyte proliferation in the intact heart. In addition, CNTF induced other pro-regenerative processes, including expression of cardioprotective genes, activation of the epicardium, enhanced intramyocardial Collagen XII deposition and leucocyte recruitment. These effects were abrogated by the concomitant inhibition of the JAK/STAT activity. Mutation of the cntf gene suppressed the proliferative response of cardiomyocytes after thoracotomy. In the regenerating zebrafish heart, CNTF injection prior to ventricular cryoinjury improved the initiation of regeneration via reduced cell apoptosis and boosted cardiomyocyte proliferation. Our findings reveal the molecular effectors of preconditioning and demonstrate that exogenous CNTF exerts beneficial regenerative effects by rendering the heart more resilient to injury and efficient in activation of the proliferative programs.

Dynamics of actinotrichia regeneration in the adult zebrafish fin.

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFß/Activin-ßA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

In vivo quantification of mechanical properties of caudal fins in adult zebrafish.

The caudal fins of adult zebrafish are supported by multiple bony rays that are laterally interconnected by soft interray tissue. Little is known about the fin's mechanical properties that influence bending in response to hydrodynamic forces during swimming. Here, we developed an experimental setup to measure the elastic properties of caudal fins in vivo by applying micro-Newton forces to obtain bending stiffness and a tensional modulus. We detected overall bending moments of 1.5×10-9-4×10-9 N m2 along the proximal-distal axis of the appendage showing a non-monotonous pattern that was not due to the geometry of the fin itself. Surgical disruption of the interray tissues along the proximal-distal axis revealed no significant changes to the overall bending stiffness, which we confirmed by determining a tensional modulus of the interray tissue. Thus, the biophysical values suggest that the flexibility of the fin during its hydrodynamic performance predominantly relies on the mechanical properties of the rays.

Influenece of the CPRmeter on angular position of elbows and generated forces during cardiopulmonary resuscitation.

OBJECTIVES: It is commonly known that ergonomics in emergency medical services (EMS) is very important. Emergency medical services workers are exposed to different conditions and they should perform a variety of tasks. MATERIAL AND METHODS: The aim of the work has been to analyze the angular position of elbows and forces generated by the upper limbs during cardiopulmonary resuscitation with and without the CPRmeter based on feedback technology. Ten male paramedics and 10 male non-paramedics, in a kneeling position, performed cardiopulmonary resuscitation (CPR) on an Ambu Megacode manikin placed on the ground. Measurements were taken after 1 min and 4 min following the beginning of the trial. The angular position of the elbows was evaluated with a BTS Smart DX 7000 motion capture system. Kistler platforms 9286BA were used for measuring forces. RESULTS: In the paramedic group, one statistically significant difference was observed in the mean difference between maximal and minimal right elbow angle in the 1st min without the device vs. the mean difference in the 4th min without the device. In the paramedic group, a 25% force decrease was observed after 4 min of resuscitation in trials without the CPRmeter in comparison to the 1st min. In trials with the CPRmeter, the force parameters were similar in the 1st and 4th min and more stable. No statistically significant differences were noticed in the control group. CONCLUSIONS: The CPRmeter has influence on the magnitude of the forces applied by the upper limbs and on the optimization of the rescuer effort during cardiopulmonary resuscitation. The CPRmeter had no influence on the position of the upper part of the kinematic chain. Int J Occup Med Environ Health 2017;30(6):909-916.

The careg element reveals a common regulation of regeneration in the zebrafish myocardium and fin.

The existence of common mechanisms regulating organ regeneration is an intriguing concept. Here we report on a regulatory element that is transiently activated during heart and fin regeneration in zebrafish. This element contains a ctgfa upstream sequence, called careg, which is induced by TGFß/Activin-ß signalling in the peri-injury zone of the myocardium and the fin mesenchyme. In addition, this reporter demarcates a primordial cardiac layer and intraray osteoblasts. Using genetic fate mapping, we show the regenerative competence of careg-expressing cells. The analysis of the heart reveals that the primordial cardiac layer is incompletely restored after cryoinjury, whereas trabecular and cortical cardiomyocytes contribute to myocardial regrowth. In regenerating fins, the activated mesenchyme of the stump gives rise to the blastema. Our findings provide evidence of a common regenerative programme in cardiomyocytes and mesenchyme that opens the possibility to further explore conserved mechanisms of the cellular plasticity in diverse vertebrate organs.

Collagen XII Contributes to Epicardial and Connective Tissues in the Zebrafish Heart during Ontogenesis and Regeneration.

Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-ß receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-ß signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart.