In the developing human musculoskeletal system, cell death with macrophage accumulation occurs in the thigh muscle and interdigital area. To comprehensively clarify the distribution of macrophages, we immunohistochemically examined 16 pairs of upper and lower extremities without the hip joint (left and right sides) obtained from 8 human fetuses at approximately 10-15 weeks of gestation. Rather than in muscles, CD68-positive macrophages were densely distributed in loose connective tissues of the flexor aspects of the extremities, especially in the wrist, hand and foot. In contrast, no or fewer macrophages were evident in the shoulder and the extensor aspects of the extremities. The macrophages were not concentrated at the enthesis of the tendon and ligament, but tended to be arranged along other connective tissue fibers. Deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed apoptosis in the hand lumbricalis muscles, but not in the area of macrophage accumulation. Likewise, podoplanin-positive lymphatic vessels were not localized to areas of macrophage accumulation. Re-organization of the connective tissue along and around the flexor tendons of the hand and foot, such as development of the bursa or tendon sheath at 10-15 weeks, might require the phagocytotic function of macrophages, although details of the mechanism remain unknown.

Purpose

We recently demonstrated the morphology of the anococcygeal ligament. As the anococcygeal ligament and raphe are often confused, the concept of the anococcygeal raphe needs to be re-examined from the perspective of fetal development, as well as in terms of adult morphology.

Materials and Methods

We examined the horizontal sections of 15 fetuses as well as adult histology. From cadavers, we obtained an almost cubic tissue mass containing the dorsal wall of the anorectum, the coccyx and the covering skin. Most sections were stained with hematoxylin and eosin or Masson-trichrome solution.

Results

The adult ligament contained both smooth and striated muscle fibers. A similar band-like structure was seen in fetuses, containing: 1) smooth muscle fibers originating from the longitudinal muscle coat of the anal canal and 2) striated muscle fibers from the external anal sphincter (EAS). However, in fetuses, the levator ani muscle did not attach to either the band or the coccyx. Along and around the anococcygeal ligament, we did not find any aponeurotic tissue with transversely oriented fibers connecting bilateral levator ani slings. Instead, in adults, a fibrous tissue mass was located at a gap between bilateral levator ani slings; this site corresponded to the dorsal side of the ligament and the EAS in the immediately deep side of the natal skin cleft.

Conclusion

We hypothesize that a classically described raphe corresponds to the specific subcutaneous tissue on the superficial or dorsal side of the anococcygeal ligament.