Development of the human Achilles tendon enthesis organ.

The attachment of the Achilles tendon is part of an 'enthesis organ' that reduces stress concentration at the hard-soft 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 effects of ageing on the distribution of vesicular acetylcholine transporter immunoreactive inputs to pelvic motoneurons of male Wistar rats.

Age-related changes in the number and size of large cholinergic terminals immunoreactive for vesicular acetylcholine transporter (VAChT), were documented for the dorsolateral nucleus (DLN), retrodorsolateral nucleus (RDLN) and spinal nucleus of the bulbospongiosus (SNB) of the lumbosacral spinal cord of male rats. The most significant changes were a large increase in the number and size of cholinergic terminals within the DLN of aged animals, together with a small decrease in terminal number within the RDLN. No significant age-associated differences in VAChT labeling were seen within the SNB. In both age groups, SNB motoneurons projecting to the levator ani muscle received about 9 to 10 contacts from large cholinergic terminals. Ultrastructural examination of the terminals revealed structures likely to be postsynaptic subsurface cisterns that are characteristic of type C terminal boutons. Since both the DLN and SNB contain motoneurons innervating pelvic muscles and sphincters, these findings provide further evidence for a central cholinergic influence on micturition and sexual reflexes and suggest that this may remain robust in the face of ageing.

Ageing reduces the number of vesicular glutamate transporter 2 containing immunoreactive inputs to identified rat pelvic motoneurons.

The immunocytochemical localisation of vesicular glutamate transporters, VGLUT1 and VGLUT2, was employed to identify putative glutamatergic axon terminals innervating pelvic motoneurons. VGLUT1 terminals were sparsely distributed within lumbosacral spinal motoneuron pools, including the dorsolateral nucleus, retrodorsolateral nucleus and spinal nucleus of the bulbospongiosus. This was in marked contrast to VGLUT2 which was expressed in a robust innervation of these areas. Retrograde tracing was used to reveal motoneurons innervating the levator ani (LA) muscle. On these neurons, associations with VGLUT2 immunoreactive terminals were abundant while those with VGLUT1 were rare. Ultrastructural investigations revealed that VGLUT2 immunoreactive terminals made asymmetric synaptic contacts with dendrites of retrogradely labelled LA motoneurons. Quantification of VGLUT2 immunoreactive boutons in close association with these dendrites was carried out in young and aged animals using light microscopy. This revealed a significant decline in the numbers of VGLUT2 immunoreactive boutons on the more distal dendrites of motoneurons in aged rats. VGLUT2 boutons were reduced by approximately 21% from 11.25+/-0.5 per 35-mum length of dendrite in young rats to 8.89+/-0.5 in aged animals. This decline in glutamatergic input may reduce the excitability of LA motoneurons and consequently decrease the capacity of the rat to induce reflexive erections.

The relationship between serotonin, dopamine beta hydroxylase and GABA immunoreactive inputs and spinal preganglionic neurones projecting to the major pelvic ganglion of Wistar rats.

Preganglionic neurones in the lumbosacral spinal cord give rise to nerves providing the parasympathetic and sympathetic innervation of pelvic organs. These neurones are modulated by neurotransmitters released both from descending supra-spinal pathways and spinal interneurones. Though serotonin has been identified as exerting a significant influence on these neurones, few studies have investigated the circuitry through which it achieves this particularly in relation to sympathetic preganglionic neurones. Using a combination of neuronal tracing and multiple immunolabeling procedures, the current study has shown that pelvic preganglionic neurones receive a sparse, and probably non-synaptic, axosomatic/proximal dendritic input from serotonin-immunoreactive terminals. This was in marked contrast to dopamine beta hydroxylase-immunoreactive terminals, which made multiple contacts. However, the demonstration of both serotonin, and dopamine beta hydroxylase immunoreactive terminals on both parasympathetic and sympathetic preganglionic neurones provides evidence for direct modulation of these cells by both serotonin and norepinephrine. Serotonin-containing terminals displaying conventional synaptic morphology were often seen to contact unlabeled somata and dendritic processes in regions surrounding the labeled preganglionic cells. It is possible that these unlabeled structures represent interneurones that might allow the serotonin containing axons to exert an indirect influence on pelvic preganglionic neurones. Since many spinal interneurones employ GABA as a primary fast acting neurotransmitter we examined the relationship between terminals that were immunoreactive for serotonin or GABA and labeled pelvic preganglionic neurones. These studies were unable to demonstrate any direct connections between serotonin and GABA terminals within the intermediolateral or sacral parasympathetic nuclei. Colocalization of serotonin and GABA was very rare but terminals immunoreactive for each were occasionally seen to contact the same unlabeled processes in close proximity. These results suggest that in the rat, the serotonin modulation of pelvic preganglionic neurones may primarily involve indirect connections via local interneurones.

Neurocalcin-alpha immunoreactivity in the enteric nervous system of young and aged rats.

The distribution of the calcium binding protein neurocalcin a has been examined in the enteric nervous system of young adult (3 months) and aged (24+ months) male rats by immunofluorescence. Neurocalcin-immunoreactive (NC-ir) neurons were observed in the submucous and myenteric plexuses throughout the gastrointestinal tract from the oesophagus to the distal large intestine. NC-ir nerve terminals were also seen on NC-ir and NC-negative neurons. Semiquantitative estimates revealed fewer NC-ir neurons in the submucous plexus than in the myenteric plexus. The greatest occurrence of NC-ir neurons was in the small and large intestine. NC-ir axons were seen in the mucosa and also in between the ganglia of the myenteric plexus. In the aged rats, there were no discernible changes in the numbers of NC-ir neurons in th e oesophagus and stomach, with an increase in the pylorus and slight decreases in the small and large intestines. No decrease in NC-ir was observed in the distal large intestine. NC-ir neurons never contained lipofuscin age pigment and many enteric neuro ns devoid of NC-ir contained age pigment. Like other previously investigated calcium-binding proteins in enteric neurons, the distribution of NC shows much variability from one part of the intestine to another. The observed slight decreases in the number of NC-ir enteric neurons in aged rats may compromise the regulation of calcium in these neurons.

Evidence for a decrease in sympathetic control of intestinal function in the aged rat.

The ability of carbachol and isoprenaline to contract and relax respectively the longitudinal layer of ileal smooth muscle has been compared in rats aged six and twenty-four months. The concentration response curve to carbachol did not vary with the age of the animal. In contrast, the ability of isoprenaline to relax longitudinal smooth muscle precontracted with carbachol was significantly (p less than 0.02) reduced in the twenty-four month age group. This reduced response was due to a decrease in the maximal relaxation induced by isoprenaline rather than by a shift to the right of its dose-response curve. These results are discussed in the context of previous histochemical and microscopical studies which have shown a marked reduction in the density of the sympathetic innervation of the rat small intestine in old age.

Age and sex do not affect the volume, cell numbers, or cell size of the suprachiasmatic nucleus of the rat: an unbiased stereological study.

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.

Quantitative analysis of the cervical sympathetic trunk in young adult and aged rats.

The cervical sympathetic trunk of rats aged 4 and 24 months was examined quantitatively with particular reference to the predominating population of unmyelinated axons by using electron microscope photomontages of transverse sections of the entire nerve trunk. No obvious neuropathological or degenerative changes were observed in animals of either age. At 24 months the mean diameter of unmyelinated axons, the mean number of axons/100 microns2 and the mean number of axons associated with individual Schwann cell units was significantly greater than in the young adult animals. With regard to the small population of myelinated axons in the nerve trunk, the thickness of the myelin sheath did not change with age. The numbers of endoneurial blood vessels in the trunk was not significantly different. This analysis of the cervical sympathetic trunk is consistent with other morphological, histochemical and biochemical evidence which suggests that elements of the cervical sympathetic system are well preserved in aged rats.

Morphometric studies on pre- and paravertebral sympathetic neurons in the rat: changes with age.

Morphometric measurements have been made on rat sympathetic neurons at ages between 6 and 24 months. In neurons of the coeliac-superior mesenteric ganglion there is a marked decrease in the neuronal packing density between 12 and 18 months which is accompanied by increases in the size of the neurons and their nuclei. In the superior cervical ganglion, no changes in packing density are seen until 18 months after which a decrease occurs, accompanied by slight increases in the neuronal parameters. These post-maturation changes occurring throughout adult life reveal a continued dynamism of sympathetic neurons into old age as well as revealing further differences between populations of sympathetic neurons.

A quantitative study of the effects of age on the noradrenergic innervation of Auerbach's plexus in the rat.

Noradrenergic nerves were demonstrated in stretch preparations of Auerbach's plexus and longitudinal muscle from the proximal jejunum of Wistar rats using glyoxylic acid-induced fluorescence. The density of the noradrenergic nerve plexus and the number of nerve terminal varicosities/frame area were assessed using a Quantimet 800 image analyser and the number of varicosities/unit length of nerve was measured manually with a calibrated planimeter. With increasing age, especially between 12 and 18 months there occurs a breakdown of plexus regularity and noticeably reduced levels of axonal fluorescence. Image analysis showed a decrease in the total area of the plexus of more than 50% and a decrease of almost 75% in the total number of varicosities. The frequency of varicosities per 100 micron of axon decreased from 18.79 at 12 months to 14.79 at 18 months. Significant changes in these parameters did not occur during the following 6 months. The dramatic decrease in the density of the noradrenergic innervation of Auerbach's plexus and the fall in number of varicosities with age implies a reduction in the potential of the sympathetic nervous system to influence control over motility of the jejunum in the aged rat.

Age related heterogeneity of lipopigments in human sympathetic ganglia.

The histochemical, autofluorescence and morphological characteristics of lipopigments accumulating with age in the human sympathetic neurons were studied. The spectral characteristics of the lipopigment autofluorescence change with age suggesting the accumulation of additional components in the residual bodies. This component is electron microscopically highly osmiophilic and stains also with silver staining. These changes may indicate the gradual melanization of the lipopigments due to auto-oxidation of catecholamines. The accumulation of the melanized form of lipopigment may be a sign of more advanced cellular trauma.

Development of a quantitative histochemical method for determination of succinate dehydrogenase activity in autonomic neurons and its application to the study of aging in the autonomic nervous system.

An accurately validated method was developed for quantitative determination of succinate dehydrogenase (EC 1.3.99.1; SDH) activity in individual sympathetic neuron perikarya by microdensitometric measurement of an SDH-nitroblue tetrazolium-derived formazan final reaction product. Optimal incubation medium and reaction conditions were determined for measurement of reaction product in cryostat sections of rat superior cervical and celiac-superior mesenteric ganglia. The Beer-Lambert laws were verified for the ganglion tissue, and microdensitometric measurements (expressed as mean cell density readings; MCDR/min-1), characteristic of the Michaelis-Menten equation, enabled the results to be used for enzyme kinetic determinations of SDH activity. Km and Vmax values were obtained following Hans linear transformation of the readings. Between the ages of 6-24 months no significant variations in Km values were recorded, indicating an unchanged structure for SDH (overall mean Km = 0.083 +/- 0.055 mM). However, in both ganglia there were significant decreases (ranging from 43-54%) in Vmax values for SDH at 24 months. The overall mean Vmax value at 6 months was 4.01 +/- 0.61 (MCDR) and at 24 months was 2.07 +/- 0.76 (MCDR). This suggests that an overall decrease in metabolic activity takes place with age in sympathetic neurons of the rat superior cervical and celiac-superior mesenteric ganglia.

Electron probe x-ray microanalysis of small granulated cells in rat sympathetic ganglia after sequential aldehyde and dichromate treatment.

Rat adrenal medulla and celiac-mesenteric sympathetic ganglia were fixed by a glutaraldehyde/formaldehyde-potassium dichromate-osmium treatment sequence and plastic-embedded. Fine sections were examined by electron probe x-ray microanalysis. Comparable peaks for chromium (Kalpha = 5.4 keV) were obtained from cytoplasmic fields containing membrane-bounded inclusion granules in both adrenomedullary noradrenaline cells and a type (type II) of sympathetic small granulated cell whose inclusion granules closely resemble those of the adrenomedullary noradrenaline cell. Chromium was not detected in granules within adrenomedullary adrenaline cells nor in two other sympathetic small granualted cell types. In no material was chromium detected in agranular cytoplasmic or nuclear fields. Since chromium binds to the Schiff monobase formed by glutaraldehyde and noradrenaline during fixation, we infer that noradrenaline is present in the granules of the type II sympathetic small granulated cell, as well as in adrenomedullary noradrenaline cells.

Age-related changes in the morphology of preganglionic neurons projecting to the paracervical ganglion of nulliparous and multiparous rats.

The aim of this study was to investigate age-related changes in preganglionic neurons in the lumbar and sacral spinal cord of the female rat that may underlie impaired control of the urogenital system in old age. Preganglionic sympathetic and parasympathetic neurons of young adult, aged nulliparous and aged multiparous rats were identified by retrograde tracing with cholera toxin subunit-B and subsequent immunocytochemistry. Labeled preganglionic neurons were scanned, processed and analyzed using the confocal microscope. Measurements were made of soma area, number of primary dendrites, number of dendritic branch points and total dendritic length. There were significant decreases in the number of primary dendrites, number of dendritic branch points and total dendritic length of sympathetic preganglionic neurons in both nulliparous and multiparous aged rats compared to the young adult group. No significant differences were found in the dendritic morphology of aged parasympathetic preganglionic neurons. Soma area was not significantly different between age groups for sympathetic or parasympathetic preganglionic neurons. These changes in dendritic morphology may result in altered control of the lower urogenital tract in aged nulliparous and multiparous female rats.

Reserpine-induced catecholamine depletion from small cells in rat sympathetic ganglia.

Two reserpine dosage schedules were applied to Wistar rats (a) 5 mg/kg i.p. 6 hr before sacrifice and (b) 5 mg/kg i.p. at 36, 24 and 12 hr prior to sacrifice. Control animals were correspondingly sham-injected. The coeliac-mesenteric ganglion complex was removed and processed either for the Falck-Hillarp fluorescence histochemical technique or fixed in glutaraldehyde followed by 3.5% potassium dichromate for a chromaffin-type reaction. After a single reserpine injection there was a statistically significant (p less than 0.001) reduction in the percentage of 'chromaffin-positive' cells and a statistically significant (p less than 0.001) increase in the percentage of 'chromaffin-negative' cells compared with controls. No obvious reduction in fluorophore emission from small intensely fluorescent (SIF) cells was observed. After prolonged reserpinization (3 x 5 mg/kg) there was a highly significant reduction in the percentage of 'chromaffin-positive' small cells and also a significantly lower (by a factor of 2) level of fluorescent emission from SIF cells. The catecholamine-releasing potential of small cells is demonstrated by these results.

Morphological evidence for the maintenance of the cervical sympathetic system in aged rats.

Elements of the cervical part of the sympathetic nervous system have been examined quantitatively in four and twenty-four month Wistar rats. The number of unmyelinated axons in the cervical trunk had significantly (P less than 0.002) increased in the old animals and the number of myelinated axons comprised less than 4% of the total number at both ages. The number of neurons in the superior cervical ganglion and the density of noradrenergic innervation of two of its target organs, the iris and the submandibular gland, were insignificantly different at the two ages. These results are consistent with other evidence which suggests that the cervical sympathetic system is maintained throughout the adult lifespan in rats.

Fluorescence histochemical evidence for decreased noradrenaline synthesis in sympathetic neurones of aged rats.

Measurements of formaldehyde-induced fluorescence of noradrenaline (NA) in sympathetic neuronal perikarya of aged (24-month) and younger (4-month) control rats revealed little or no NA fluorescence in many sympathetic neurones of aged rats: neuronal fluorescence in younger rats was substantially greater. Monoamine oxidase inhibition increased NA fluorescence in all rats, but the increase was greater in the 4-month controls. Reserpine administration caused an almost complete disappearance of NA fluorescence after 12 h. However, normal NA levels were restored in the 4-month controls after 48 h but not for 7 days in the aged rats.

Long-lasting effects of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on prevertebral sympathetic neurones in the rat.

Following intraperitoneal administration of the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) in rats, the irides and pieces of jejunum were treated by the glyoxylic acid method in order to reveal their adrenergic nerves. Seven days after the injection, the fluorescence of adrenergic nerves in the iris was partly restored and by 35 days was indistinguishable in density and brightness from controls. In contrast, even 118 days post-injection, the adrenergic component of Auerbach's plexus of the jejunum was still markedly affected by the DSP-4. The regeneration was incomplete, giving a patchy appearance, and less fluorescent than the controls. It is suggested that, when injected intraperitoneally, DSP-4 can have more potent effects than hitherto realised.

Decrease in synapsin I staining in the hypogastric ganglion of aged rats.

The rat hypogastric ganglion (HG) contains populations of both sympathetic and parasympathetic postganglionic neurons which supply the lower pelvic viscera. These neuron populations can be identified by tyrosine hydroxylase (TH) and NADPH-diaphorase (NADPH-d) staining, respectively. The effects of age on the distribution of synapsin I, a nerve terminal marker, in relation to these neuron populations has been investigated in young adult and aged rats. Most synapsin staining was axosomatic and was markedly reduced in the aged animals particularly in relation to sympathetic (NADPH-d-negative/TH-positive) neurons. Image analysis of synapsin I staining in relation to individual sympathetic neurons confirmed that there was a reduction with age of about 50% but no change in synapsin I staining in relation to parasympathetic neurons. These results suggest that synaptic transmission and peripheral integration may be affected in old age and that the autonomic control of the pelvic viscera may be compromised as a result, particularly with regard to the sympathetic innervation. Other autonomic ganglia were also studied for comparison but no such age-related differences were observed.

Total neuronal numbers of rat lumbosacral primary afferent neurons do not change with age.

Total cell numbers and neuronal diameters of L6 and S1 dorsal root ganglia, which provide a sensory innervation to pelvic viscera were determined in young adult (3-months-old) and compared to those in old (24-months-old) male rats. Two methods of cell counting, serial (section) reconstructions and total profile counting, were used in this study. Our data showed that the total number of L6 and S1 dorsal root ganglia (DRG) cells and their diameters remain essentially constant from 3 to 24 months of age. These results have shown that rat DRG cell numbers do not change during adult life and that neurogenesis of DRG cells in adult rats or neuronal cell death in aged rats cannot be supported. These findings are also consistent with other data supporting the maintenance of pelvic sensory innervation in old age.