Previous studies on ageing animal and human subjects have demonstrated a significant overall decline in neuronal numbers in the myenteric plexus of the enteric nervous system (ENS). Our study aimed to confirm this observation by counting myenteric neurons stained with the panneuronal markers PGP 9.5 and NADHdiaphorase. We also wished to examine the possibility that particular subpopulations of neurons are vulnerable. Therefore, we have immunostained and counted a number of nerve cell groups within the myenteric plexus of old and young Sprague Dawley rats using markers which reflect some of the neuronal phenotypes present, including ChAT and VIP. The number of neurons demonstrating NADH-diaphorase activity was significantly reduced (P 0.05) by approximately 15 % in old rats. However, the number of neurons stained for PGP 9.5 immunohistochemistry was not reduced and demonstrated larger numbers of neurons than the NADH-diaphorase method. None of the other neuronal markers studied showed any significant reductions with age. In contrast to previous work, this study has gathered little evidence for extensive cell loss in the myenteric plexus of the aged rat, either in overall populations, or in any of the principal functional groups of neurons.
The attachment of the Achilles tendon is part of an 'enthesis organ' that reduces stress concentration at the hardsoft tissue interface. The organ also includes opposing sesamoid and periosteal fibrocartilages, a bursa and Kager's fat pad. In addition, the deep crural and plantar fasciae contribute to Achilles stress dissipation and could also be regarded as components. Here we describe the sequence in which these various tissues differentiate. Serial sections of feet from spontaneously aborted foetuses (crown rump lengths 22-322 mm) were examined. All slides formed part of an existing collection of histologically sectioned embryological material, obtained under Spanish law and housed in the Universidad Complutense, Madrid. From the earliest stages, it was evident that the Achilles tendon and plantar fascia had a mutual attachment to the calcaneal perichondrium. The first components of the enthesis organ to appear (in the 45-mm foetus) were the retrocalcaneal bursa and the crural fascia. The former developed by cavitation within the mesenchyme that later gave rise to Kager's fat pad. The tip of the putative fat pad protruded into the developing bursa in the 110-mm foetus and fully differentiated adipocytes were apparent in the 17-mm foetus. All three fibrocartilages were first recognisable in the 332-mm foetus -at which time adipogenesis had commenced in the heel fat pad. The sequence in which the various elements became apparent suggests that bursal formation and the appearance of the crural fascia may be necessary to facilitate the foot movements that subsequently lead to fibrocartilage differentiation. The later commencement of adipogenesis in the heel than in Kager's pad probably reflects the non-weight environment in utero. The direct continuity between plantar fascia and Achilles tendon that is characteristic of the adult reflects the initial attachment of both structures to the calcaneal perichondrium rather than to the skeletal anlagen itself.
The circadian rhythms displayed by numerous biological functions are known to be sex specific and affected by aging. It has not been settled yet whether the sex- and age-related characteristics of circadian rhythms derive from changes in the anatomy of the suprachiasmatic nucleus. To shed light on these issues, we applied unbiased stereological techniques to estimate the volume of the suprachiasmatic nucleus as well as the total number of its cells and the mean volume of their somata and nuclei in progressively older groups of male and female Wistar rats (aged 1, 6, 12, 18, 24, and 30 months). The volume of the nucleus was estimated with the Cavalieri principle on serial sections. The total numbers of neurons and astrocytes were estimated by applying the optical fractionator, and the mean somatic and nuclear volumes of cells were estimated by using isotropic, uniform random sections and the nucleator method. On average, the volume of the suprachiasmatic nucleus was 0.044 mm3, and the total number of neurons and astrocytes was 17,400. Cells of the dorsomedial and ventrolateral components of the nucleus, which are morphologically different, have identical mean perikaryal and nuclear volumes, which we estimated to be 750 microns3 and 400 microns3, respectively. We further demonstrated that, at all ages analysed, the volume of the suprachiasmatic nucleus, the total cell number, and the mean somatic and nuclear volumes of its cells are affected neither by the age nor by the sex of the animal, regardless of the presence of sex- and age-related variations in circadian rhythms. However, the possibility that females may display changes in the volume of the suprachiasmatic nucleus at older ages cannot be ruled out. No effect of aging was observed in the total number of neurons or in the total number of astrocytes.
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