Regenerative Limb Research Articles

Adaptation of Glucose Metabolism to Limb Autotomy and Regeneration in the Chinese Mitten Crab.

Limb autotomy and regeneration represent distinctive responses of crustaceans to environmental stress. Glucose metabolism plays a pivotal role in energy generation for tissue development and regeneration across various species. However, the relationship between glucose metabolism and tissue regeneration in crustaceans remains elusive. Therefore, this study is aimed at analyzing the alterations of glucose metabolic profile during limb autotomy and regeneration in Eriocheir sinensis, while also evaluating the effects of carbohydrate supplementation on limb regeneration. The results demonstrated that limb autotomy triggered a metabolic profile adaption at the early stage of regeneration. Hemolymph glucose levels were elevated, and multiple glucose catabolic pathways were enhanced in the hepatopancreas. Additionally, glucose and ATP levels in the regenerative limb were upregulated, along with increased expression of glucose transporters. Furthermore, the gene expression and activity of enzymes involved in gluconeogenesis were repressed in the hepatopancreas. These findings indicate that limb regeneration triggers metabolic profile adaptations to meet the elevated energy requirements. Moreover, the study observed that supplementation with corn starch enhanced limb regeneration capacity by promoting wound healing and blastema growth. Interestingly, dietary carbohydrate addition influenced limb regeneration by stimulating gluconeogenesis rather than glycolysis in the regenerative limb. Thus, these results underscore the adaptation of glucose metabolism during limb autotomy and regeneration, highlighting its essential role in the limb regeneration process of E. sinensis.

Hedgehog signaling is essential in the regulation of limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds-HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells.

Regenerative Vs Flap-Based Limb Salvage: A Multi-Centered, Prospective, Randomized Controlled Trial

Aim: The goal of this study was to compare success rates of a regenerative limb salvage approach (rLS) using dehydrated human chorion amnion membrane (dHACM) to traditional flap-based limb salvage (fLS). Materials & methods: This prospective RTC enrolled patients presenting with complex extremity wounds over a 3-year period. Primary outcomes included success of primary reconstruction, persistence of exposed structures, time to definitive closure, and time to weight bearing. Results: Patients meeting inclusion criteria were randomized to fLS (n=14) or rLS (n=25). The primary reconstructive method was successful for 85.7% of fLS subjects and 80% of rLS subjects (p=1.00). Conclusion: This trial provides strong evidence that rLS is an effective option in the setting of complex extremity wounds, with success rates comparable to traditional flaps. Clinical Trial Registration: NCT03521258 (ClinicalTrials.gov).

Expression and function of WNT4 involved in larvae development and limb regeneration in Portunus trituberculatus

The wingless-related integration site (WNT) proteins are a family of secreted glycoproteins that are evolutionarily conserved and are believed to be involved in evolution in vertebrates and invertebrates. WNT signaling pathways may be associated with limb regeneration and development in crustaceans. However, the detail mechanisms remain unclear. Therefore, the distribution of WNT4 in the hepatopancreasmuscle, hemocyte, ganglion, heart, eyestalk, gill tissue, and different larvae development stages of the swimming crab (Portunus trituberculatus) were characterized using immunofluorescence, real-time PCR, and Western blotting. Significant PtWNT4 expression was detected in heart and eyestalk. In addition-PtWNT4 was expressed in all larval stages of P. trituberculatus with a dynamic expression pattern, especially in the eyestalk and other organs in the carapace area. The injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels in the eyestalk, heart, and muscle, resulting in 1.9-fold, 2.2-fold, and 2.7-fold decreases compared with those detected in the group injected with crab saline (P<0.05), respectively, indicating successful gene silencing. Overall, expression analysis on the WNT4 using RNAi provides an insight to its functional mechanism during limb regeneration in P. trituberculatus. The results not only demonstrated the requirement for WNT4 in limb regeneration of Crustaceans, but also suggested its ability to promote larval development at specific stages.

Regenerative Limb Salvage: A Novel Technique for Soft Tissue Reconstruction of Pediatric Extremities

AbstractBackground In complex extremity wounds, free flap-based limb salvage (fLS) is the standard of care. However, fLS is resource- and cost-intensive, and the limited availability of pediatric microsurgical expertise exacerbates these challenges. Regenerative LS (rLS) addresses these barriers to care. The aim of this study was to quantify the efficacy, safety, and cost-effectiveness of rLS in complex pediatric extremity wounds.Methods We conducted a retrospective cohort study of pediatric LS at a single hospital. Subjects were treated with fLS or rLS based on surgeon preference. Primary outcome measures were: definitive wound closure rates and time, rates of return to ambulation, number and length of procedures to achieve definitive closure, and rates of perioperative complications. Statistical analyses were performed utilizing the Wilcoxon Mann–Whitney U test with statistical significance set at p &lt; 0.05.Results Over a 2-year period, nine consecutive patients presented with complex extremity wounds requiring fLS (n = 4) or rLS (n = 5). Demographics and wound characteristics were similar between groups. Compared with fLS, the rLS group achieved definitive wound closure 85.8% more quickly (13.8 vs. 97.5 days, p &lt; 0.02), required 64.5% less operative time (132.6 vs. 373.0 minutes, p &lt; 0.02), had fewer perioperative complications (0 vs. 5), and required fewer intensive care unit stays (0 vs. 1.3 days).Conclusion These data indicate that rLS is a safe and efficacious option in pediatric patients requiring soft tissue reconstruction for LS. Use of this novel technique can restore the reconstructive ladder, thereby expand patient access to pediatric LS while minimizing morbidity and reducing LS-related resource utilization.

Suppression of limb regeneration by RNA interference of WNT4 in the swimming crab Portunus trituberculatus.

The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p <0.05). Expression analysis on the WNT4 using RNAi provide important information for understanding its functional mechanism during limb regeneration in P. trituberculatus. The results also contribute to our understanding of the role of Wnt signalling during limb regeneration in crustaceans.

UP‐REGULATION OF TFAP2C BY FGF DURING SHH INDUCTION IN LIMB BUDS

During development, limb outgrowth has been linked to a reciprocal feedback loop between Fibroblast growth factors (Fgfs) and Sonic Hedgehog (Shh). Fgfs from the apical ectodermal ridge (AER) at the distal tip of the limb bud promote secretion of Shh from the zone of polarizing activity (ZPA), a cluster of cells in the distal posterior aspect of limb bud mesoderm. In turn, Shh induces Fgf from the posterior AER. This reciprocal loop is also critical to limb regeneration, and previous studies have shown that Shh up‐regulation correlates with the capacity of Fgfs to accomplish regenerative limb outgrowth. Using gene arrays, we have identified a number of potential Fgf downstream targets in the Fgf‐Shh pathway. In this report, we examined the expression of Transcription factor AP‐2 gamma (Tfap2c).To determine the time course of Fgf‐induced Tfap2c up‐regulation, we implanted Fgf‐soaked beads into stage 23 chick wing buds and evaluated Tfap2c expression by in situ hybridization at several time points.Tfap2c was up‐regulated within 3 hours of Fgf exposure; however, treatment with the protein synthesis inhibitor cycloheximide abolished induction.We conclude that Tfap2c is a close, but indirect, downstream target of Fgf during limb development and regeneration. Further studies are needed to determine whether Tfap2c participates in the Fgf‐Shh pathway or plays an alternative role in limb outgrowth.Grant Funding Source: departmental

Effect of ammodytin L from the venom of Vipera ammodytes on xenopus laevis differentiated muscle fibres and regenerating limbs

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [ 3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters

Adult urodele amphibians can regenerate their limbs and tail. Based on their roles in other developing systems, Hox genes are strong candidates for genes that play a role in regulating pattern formation during regeneration. There are four homologous clusters of Hox genes in vertebrate genomes. We isolated cDNA clones of two newt homeobox genes from homologous positions within two Hox clusters; Hox-4.5 and Hox-3.6. We used RNase protection on nonamputated (normal) and regenerating newt appendages and tissue to compare their transcriptional patterns. Both genes show increased expression upon amputation with similar kinetics. Hox-4.5 and Hox-3.6 transcription is limited to the mesenchymal cells in the regenerates and is not found in the epithelial tissue. In addition to regenerating appendages, both genes are transcriptionally active in adult kidney of the newt. Striking differences were found in the regulation of Hox-4.5 and Hox-3.6 when they were compared in unamputated limbs and in regenerating forelimbs versus regenerating hindlimbs. Hox-4.5 is expressed in the blastema of regenerating fore- and hindlimbs, but Hox-4.5 transcripts are not detectable in normal limbs. In contrast, Hox-3.6 transcripts are found exclusively in posterior appendages, but are present in normal as well as regenerating hindlimbs and tails. Hox-4.5 is also expressed at a higher level in proximal (mid-humerus) regenerates than in distal ones (mid-radius). When we proximalized the positional memory of a distal blastema with retinoic acid, we find that the early expression level of Hox-4.5 is also proximalized. When the expression of these genes is compared to the expression of two previously reported newt Hox genes, a consistent pattern emerges, which can be interpreted in terms of differential roles for the different Hox clusters in determining regenerative limb morphology.

Limb Development and Regeneration

Experiments on developing and regenerating vertebrate limbs have led to the idea that pattern formation and growth control are causally linked. The mechanism by which position-specific growth occurs is termed intercalation, and evidence is presented that implicates intercalation in the initiation, maintenance and cessation of growth during limb formation. We conclude that among the variety of cell types present in limbs, only fibroblasts have been shown to possess the positional information necessary for intercalation. Hence we propose that the limb pattern is generated by intercalation between fibroblasts to give rise to a connective tissue scaffold, which in turn dictates the positioning and morphogenesis of all of the differentiated cell types of the limb. Finally, we review evidence that regenerative failure among higher vertebrates is linked to defects in the intrinsic cellular mechanisms of growth control (intercalation) and conclude that progress towards the goal of stimulating regenerative limb outgrowth in non-regenerating vertebrates will be contingent upon a better understanding of these intrinsic mechanisms.

Effects of two dithiocarbamates on the grass shrimp, Palaemonetes pugio: molt-related toxicity and inhibition of limb regeneration.

The effects of two dithiocarbamate formulations (Aquatreat® DNM-30 and Busan® 85) on the initiation and progression of limb regeneration following the removal of the left fifth walking leg (pereiopod) were studied with the estuarine grass shrimp,Palaemonetes pugio. Exposure to either Aquatreat DNM-30 or Busan 85 affected both the initiation and progression of limb regeneration; the shrimp exhibited dose-related inhibition of regenerative growth. The resultant EC50 values for inhibition of regenerative limb growth were well below the median lethal concentrations (LC50s) for grass shrimp. In tests with molting grass shrimp, Busan 85 was more acutely toxic than Aquatreat DNM-30.

Effects of ecdysterone, inokosterone and eyestalk ablation on limb regeneration in the fiddler crab, Uca pugilator

AbstractBilateral ablation of eyestalks or injection of either ecdysterone or inokosterone (two 25‐μg doses or five 10‐μg doses into crabs weighing on an average 1.5 g) caused precocious proecdysis and ecdysis in the fiddler crab, Uca pugilator. On a weight basis ecdysterone was more effective than inokosterone in exerting the effects. Whether the accelerated proecdysis was accompanied by regenerative limb growth depended on the degree of limb bud development at the time of treatment with ecdysones. Ecdysterone and inokosterone blocked the initiation of basal limb growth when administered as two 25‐μg doses into crabs exhibiting scar tissue without any visible limb bud. When injected into crabs with limb buds in various stages of basal limb growth exogenous ecdysones accelerated basal limb growth and led to proecdysial limb growth. The degree of acceleration of limb bud growth noticed within three to six days after injection of ecdysones into crabs with limb buds in early stages of basal limb growth was greater than that resulting from eyestalk ablation. In crabs with limb buds in later stages of basal growth, the degree of acceleration of limb bud growth evoked by exogenous ecdysones was similar to that resulting from eyestalk ablation. The final sizes of the limb buds preceding ecdysis in eyestalkless crabs and crabs injected with ecdysones depended on the proecdysial duration available for regeneration.The rate of proecdysial limb growth has been known to vary with the speed at which the crabs complete the proecdysial stages of the molt cycle. This study shows that the time required for the initiation and completion of the basal limb growth also varies depending on how soon the intact and eyestalkless crabs approach proecdysis and ecdysis.

Comparison of the abilities of ?-ecdysone and 20-hydroxyecdysone to induce precocious proecdysis and ecdysis in the fiddler crab, Uca pugilator

1. The effects of single, double or multiple injections of α-ecdysone and 20-hydroxyecdysone on molting and limb regeneration in the fiddler crab, Uca pugilator, were determined. 2. A single injection of 12 μg of 20-hydroxyecdysone failed to initiate proecdysis in crabs weighing 1.2 to 1.6 g. Administration of 12, 5, and 5 μg of 20-hydroxyecdysone on Days 0, 3, and 6 respectively induced precocious initiation of proecdysis but not precocious ecdysis. However, when two large dosages (20 or 25 μg) of 20-hydroxyecdysone were injected within a period of one or two days all the crabs completed proecdysis and initiated ecdysis precociously but failed to complete the ecdysis and died. 3. Administration of 25 μg of α-ecdysone as a single injection failed to initiate precocious proecdysis. However, when two injections of 25 μg each were given 36% of the crabs completed proecdysis and successfully molted precociously. Injection of larger dosages (80 μg α-ecdysone either as four 20 μg injections or two 40 μg injections) induced precocious proecdysis and ecdysis in 75–86% of the crabs. In contrast to the crabs which died during ecdysis after receiving high dosages of 20-hydroxyecdysone, the recipients of α-ecdysone underwent successful ecdysis and their postecdysial survival was normal. 4. The response of the regenerating limb buds depended on the dosage of the ecdysones administered. Dosages of 20-hydroxyecdysone which failed to initiate proecdysis promoted basal limb growth. Stronger dosages of 20-hydroxyecdysone and α-ecdysone which induced precocious proecdysis and ecdysis accelerated limb growth and caused proecdysial limb growth. The slope of the growth curve obtained for the crabs injected with high dosages of ecdysones was much steeper than that for eyestalkless crabs. 5. The interrelationship between regenerative limb growth and proecdysis and the possible role of ecdysones in the control of proecdysial events in crustaceans are discussed.