Pro and Con: How Important Is the Exact Location of Adductor Canal and Femoral Triangle Blocks?

In this Pro-Con commentary article, we debate the importance of anterior thigh block locations for analgesia following total knee arthroplasty. The debate is based on the current literature, our understanding of the relevant anatomy, and a clinical perspective. We review the anatomy of the different fascial compartments, the course of different nerves with respect to the fascia, and the anatomy of the nerve supply to the knee joint. The Pro side of the debate supports the view that more distal block locations in the anterior thigh increase the risk of excluding the medial and intermediate cutaneous nerves of the thigh and the nerve to the vastus medialis, while increasing the risk of spread to the popliteal fossa, making distal femoral triangle block the preferred location. The Con side of the debate adopts the view that while the exact location of local anesthetic injection appears anatomically important, it has not been proven to be clinically relevant.

The Importance of the Saphenous Nerve in Ankle Surgery.

BACKGROUND: Recent evidence suggests that the saphenous nerve may be involved in the innervation of deeper structures at the medial ankle. In this study, we sought to determine the consistency and variability of the saphenous nerve innervation at the distal tibia and medial ankle joint capsule. METHODS: One hundred three lower extremities from 52 embalmed cadavers were dissected to identify the deep branches of saphenous nerve along its distal course. RESULTS: In all specimens, the saphenous nerve had branches, emerging between 3.9 and 8.2 cm above the medial malleolus, to the periosteum of the distal tibia and the medial capsule of the ankle joint. CONCLUSIONS: Deep branches of the saphenous nerve innervate the periosteum of the distal tibia and talocrural capsule.

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

Exploring the effects of gene dosage on mandible shape in mice as a model for studying the genetic basis of natural variation.

Mandible shape in the mouse is a complex trait that is influenced by many genetic factors. However, little is known about the action of single genes on adult mandible shape so far, since most developmentally relevant genes are already required during embryogenesis, i.e., knockouts lead to embryonic death or severe deformations, before the mandible is fully formed. We employ here a geometric morphometric approach to identify subtle phenotypic differences caused by dosage effects of candidate genes. We use mouse strains with specific gene modifications (knockouts and knockins) to compare heterozygous animals with controls from the same stock, which is expected to be equivalent to a change of gene expression of the respective locus. Such differences in expression level are also likely to occur as part of the natural variation. We focus on Bmp pathway genes (Bmp4, its antagonist Noggin, and combinations of Bmp5-7 genotypes), but include also two other developmental control genes suspected to affect mandible development in some way (Egfr and Irf6). In addition, we study the effects of Hoxd13, as well as an extracellular matrix constituent (Col2a1). We find that subtle but significant shape differences are caused by differences in gene dosage of several of these genes. The changes seen for Bmp4 and Noggin are partially compatible with the action of these genes known from birds and fish. We find significant shape changes also for Hoxd13, although this gene has so far only been implicated in skeletal patterning processes of the limbs. Comparing the effect sizes of gene dosage changes to the variation found in natural populations of mice as well as quantitative trait loci (QTL) effects on mandible shape, we find that the effect sizes caused by gene dosage changes are at the lower end of the spectrum of natural variation, but larger than the average additive effects found in QTL studies. We conclude that studying gene dosage effects have the potential to provide new insights into aspects of craniofacial development, variation, and evolution.

Site-specific cleavage of BMP4 by furin, PC6, and PC7.

Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin- and PC6-selective inhibitor alpha(1)-PDX. By contrast, alpha(1)-PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to alpha(1)-PDX. Consistent with this prediction, an alpha(1)-PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion.

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain.

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.

Proprotein convertase genes in Xenopus development.

Proprotein convertases (PCs) are a family of serine endoproteases that proteolytically activate many precursor proteins within various secretory pathway compartments. Loss-of-function studies have demonstrated a critical role for these proteases in embryonic patterning and adult homeostasis, yet little is known about how substrate selectivity is achieved. We have identified Xenopus orthologs of three PCs: furin, PC6, and PC4. In addition to previously described isoforms of PC6 and furin, four novel splice isoforms of PC6, which are predicted to encode constitutively secreted proteases, and a putative transmembrane isoform of PC4 were identified. Furin and PC6 are expressed in dynamic, tissue-specific patterns throughout embryogenesis, whereas PC4 transcripts are restricted primarily to germ cells and brain in adult frogs.