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.

Adipose tissue at entheses: the innervation and cell composition of the retromalleolar fat pad associated with the rat Achilles tendon.

This study set out to determine whether the fat pad at the attachment of the Achilles tendon has features enabling it to function as an immune organ and a mechanosensory device, and to be a source of pain in insertional tendon injuries. Sections for histology and immunohistochemistry were cut from the Achilles tendon enthesis organ of 1 day old, 1 month, 4 month and 24 month old rats. For fluorescence and peroxidase immunohistochemistry, cryosections were labelled with primary antibodies directed against PGP9.5, substance P, neurofilament 200, calcitonin gene related peptide, CD68, CD36, myeloid related protein 14, actin and vinculin. The fat pad contained not only adipocytes, but also fibrous tissue, mast cells, macrophages, fibroblasts and occasional fibrocartilage cells. It was richly innervated with nerve fibres, some of which were likely to be nociceptive, and others mechanoreceptive (myelinated fibres, immunoreactive for neurofilament 200). The fibres lay between individual fat cells and in association with blood vessels. In marked contrast, the enthesis itself and all other components of the enthesis organ were aneural at all ages. The presence of putative mechanoreceptive and nociceptive nerve endings between individual fat cells supports the hypothesis that the fat pad has a proprioceptive role monitoring changes in the insertional angle of the Achilles tendon and that it may be a source of pain in tendon injuries. The abundance of macrophages suggests that the adipose tissue could have a role in combating infection and/or removing debris from the retrocalcaneal bursa.

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 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.

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.

Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons.

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.

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.

Decreased neurocalcin immunoreactivity in sympathetic and parasympathetic neurons of the major pelvic ganglion in aged rats.

In the rat the majority of sympathetic and parasympathetic postganglionic neurons that innervate the pelvic viscera are located together in the major pelvic ganglia. We have ascertained that it is only the sympathetic population of this ganglion that exhibits age-associated attrition. Recent immunohistochemical investigations of the distribution of calcium binding proteins in this ganglion in young adult and aged rats have demonstrated that calbindin-D28k is only present in the sympathetic neurons and that the number of calbindin-immunoreactive sympathetic neurons of the aged ganglion was dramatically reduced. In the present study we have investigated the distribution of neurocalcin (NC) alpha isoform in the major pelvic ganglion. In young adults 98.7% of sympathetic neurons (identified by anti-tyrosine hydroxylase immunostaining) and 98% of parasympathetic neurons (identified by anti-nitric oxide synthase immunostaining) contained NC immunoreactivity and these figures are reduced to 68 and 45.5% in the aged group. Thus, unlike calbindin-D28k, NC is not confined to the sympathetic neuron population in the major pelvic ganglion and decreases significantly in old age in both neuronal populations. The likely effects are to impair intracellular calcium-dependent signalling in neurons of the major pelvic ganglion, possibly compounding the effects of the previously reported decrease in calbindin-D28k in the sympathetic population.

Distribution and changes with age of nitric oxide synthase-immunoreactive nerves of the rat urinary bladder, ureter and in lumbosacral sensory neurons.

In the distal parts of the urinary tract, nerves containing nitric oxide (NO) are either postganglionic parasympathetic nerves, with cell bodies in the major pelvic ganglia, or sensory nerves with cell bodies in the lumbosacral dorsal root ganglia. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive for neuronal nitric oxide synthase (NOS) in the urinary bladder, distal ureter and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made of NOS fibres innervating the dome, body and base of the urinary bladder and distal ureter. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for NOS. The dome and the body regions, in both age groups, contained no NOS-immunoreactive axons. The bladder base and distal ureter in young adults showed sparse to moderate numbers of fibres immunoreactive to NOS within the urothelium and in the subepithelium and muscle coat. In the aged rat there were slight reductions in the densities of NOS-immunoreactive nerves in all three regions. In the lumbosacral dorsal root ganglia, the percentage of NOS-immunoreactive neuronal profiles showed a significant reduction from 4.6 +/- 0.2% in young adult to 2.7 +/- 0.2% (means +/- S.E.M) in aged rats. These findings suggest that the effects of NO on the bladder and distal ureteric musculature and also its expression in dorsal root ganglion neurons are affected in aged rats and that the micturition reflex may be perturbed as a result.

Restricted diet rescues rat enteric motor neurones from age related cell death.

BACKGROUND: Alone among autonomic neurones, enteric neurones are known to be vulnerable to age related cell death; over 50% may be lost in aging rodents. A previous study demonstrated unexpectedly that neurones of the myenteric plexus from rats fed a restricted diet appeared not to suffer from extensive cell death in contrast with previous studies of ad libitum fed animals. AIMS: To compare myenteric neurone numbers in the ileum of young and aging male Sprague-Dawley rats fed either ad libitum or a restricted diet. METHODS: Neurones were counted in whole mount preparations of rat ileum stained immunohistochemically for the pan-neuronal marker PGP9.5, for choline acetyltransferase, or for nitric oxide synthase, or with NADH or NADPH histochemistry. RESULTS: Neurone numbers in the rat myenteric plexus were substantially affected by the dietary regimen: ad libitum feeding (50-60 g per day of standard rat chow) resulted in the death of about 50% of myenteric neurones in 24 month Sprague-Dawley rats, while numbers were unchanged when the daily dietary intake was halved between the ages of six and 24 months. Animals fed a double restricted diet (15 g per day) showed no cell loss at 30 months, as well as the predicted increase in longevity. Neurone loss was largely complete by 16 months in ad libitum fed animals. Numbers of cholinergic (possibly motor) neurones, as demonstrated by choline acetyltransferase immunohistochemistry, were substantially reduced in ad libitum fed aging rats but not in animals fed a restricted diet. Loss of cholinergic neurones after ad libitum feeding was confirmed by reduced numbers of neurones of a size range matching that of cholinergic neurones. CONCLUSIONS: Ad libitum feeding of adult rats has adverse effects on the survival of myenteric neurones, neurone loss commencing before 16 months of age. Cholinergic neurones appear to be particularly vulnerable to the effects of diet. Restricting dietary intake from six months of age prevents neurone loss almost entirely up to 30 months of age in these rats.

Decreased calbindin-D28k immunoreactivity in aged rat sympathetic pelvic ganglionic neurons.

The rat major pelvic ganglion contains the majority of sympathetic and parasympathetic postganglionic neurons that innervate the pelvic viscera. Previous studies have indicated that it is only the sympathetic population of this ganglion that is susceptible to age-associated changes. We have examined the distribution of the neuronal calcium binding proteins calbindin-D28k, calretinin and parvalbumin by immunohistochemistry in young adult and aged rats and have discovered that calbindin-D28k is only present in the sympathetic neurons (identified by tyrosine hydroxylase immunostaining) and not in parasympathetic neurons (identified by VIP immunostaining). In the aged rats the number of calbindin-immunoreactive sympathetic neurons of the major pelvic ganglion was dramatically reduced. Calretinin and parvalbumin-immunoreactivity was present at a lower level of fluorescence than that of calbindin immunoreactivity in all the neurons of the major pelvic ganglion and this level was unchanged in aged rats. Thus we suggest that the decline of intracellular calbindin D28k levels may lead to impaired calcium buffering capacity which might be a contributory factor in the age-associated attrition of pelvic sympathetic neurons.

Quantitative studies on myelinated and unmyelinated nerve fibres in the interatrial septal region of aged rat hearts.

Intracardiac nerve fibres from the interatrial septum were studied quantitatively and qualitatively by electron microscopy of transversely sectioned nerve bundles in male Wistar rats of 4 and 24 months. No significant changes were found in the myelinated fibre diameters, myelinated axon diameters, myelin sheath thicknesses, g ratios, myelinated fibre areas, unmyelinated axon diameters and unmyelinated axon areas. However, there was evidence of structural changes to the nerve fibres and Schwann cells at 4 and 24 months, increasing in prevalence with age: some myelinated fibres showed infolds, disruptions and clefts of the myelin sheath and accumulation of electron dense myelin-like fragments in the axoplasm. Unmyelinated axons showed fewer changes in structure but also contained similar fragments in the axoplasm. The numbers of neurotubules and neurofilaments per microm2 in unmyelinated intracardiac axons was significantly greater than in those in samples of the cervical vagal trunk. This may be an adaptation to the continuous mechanical stress experienced by these intracardiac nerves. It is concluded that there is little structural evidence to suggest that the conductive properties of intracardiac nerve fibres are adversely affected in aged rats.

The effects of age on the overall population and on sub-populations of myenteric neurons in the rat small intestine.

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 NADH-diaphorase. 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.

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.

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

The hypogastric and thirteenth thoracic ganglia of the rat: effects of age on the neurons and their extracellular environment.

Morphometric analyses of the neurons and microvessels of perfusion-fixed hypogastric (HG) and 13th thoracic (T13) ganglia have been performed in male Wistar rats aged 4, 24 and 30 mo. Estimations of HG volume employing the Cavalieri principle have also been performed and showed that the size of the aged HG is increased by 42%. Routine histological staining of the ganglia with Masson's trichrome indicated that this may be due to the increased amount of interstitial connective tissue which was apparent in the aged animals. The number of neurons per unit area progressively decreased by 38% between ages 4 and 24 mo and by 16% between ages 24 and 30 mo in the HG and by 25% (4 and 24 mo) and 2% (24 and 30 mo) in the T13 ganglion. The total number of neurons in the HG however, estimated by a physical disector analysis, was constant with age. The number of microvessels per unit area, microvessel diameter, neuronal and nuclear areas did not differ significantly between the 3 age groups studied. This observed increase in ganglionic volume and decrease in neuronal packing density may be associated with changes in the extracellular matrix, in particular in glycosaminoglycans whose presence was indicated by metachromasia of the ganglia with toluidine blue. The extracellular matrix was therefore characterised using a panel of monoclonal antibodies against glycosaminoglycans and laminin. Chondroitin-6 sulphate and chondroitin-4 sulphate were present in the interstitial connective tissue, and there was an increase in the expression of both these epitopes at 24 mo, noteably surrounding neuron cell bodies. The expression of chondroitin-4 sulphate/dermatan sulphate was unchanged, thus implying a decreased expression of dermatan sulphate with age. Keratan sulphate and the native chondroitin sulphate epitopes were absent from the ganglia at both ages. Laminin expression was increased in the aged ganglia. It is therefore clear that the constituents of the extracellular matrix are not constant throughout the adult lifespan and that the extracellular matrix may influence neuronal survival in old age. This is the first report characterising age-related changes in the extracellular matrix of autonomic ganglia.

Vagal nuclei in the medulla oblongata: structure and activity are maintained in aged rats.

The neurones and microvessels of the dorsal motor nucleus of the vagus (DMN), the nucleus ambiguus (NA) and the nucleus tractus solitarius (NTS) of 4, 24 and 30 month male Wistar rats have been examined morphometrically and by quantitative enzyme histochemical methods (4 and 24 months only) to assess the affects of old age on the structure and activity of their neurones. DMN and NTS neuronal soma area increased whilst NA neuronal area was reduced in the aged groups; the changes in neuronal size were reflected in the density of neurones per unit area. The mean diameter and percentage area occupied by microvessels was unchanged with increased age in all three nuclei. Quantitative assessment of cytochrome oxidase and NADH-tetrazolium reductase activities in the nuclei revealed no changes in old age, indicating that old age does not affect neuronal or metabolic activity of central vagal neurones. These results compliment previous age-related studies on the vagus nerve and nodose ganglion in which little change has been reported, suggesting that the vagal system is well preserved in aged rats.

Neurons and microvessels of the nodose (vagal sensory) ganglion in young adult and aged rats: morphometric and enzyme histochemical studies.

The neuron cell bodies and microvessels in sections of the nodose (vagal sensory) ganglion (NG) of Wistar rats of 4- and 24-months of age have been examined morphometrically and by quantitative enzyme histochemistry. The range of neuronal somata areas was similar at the two ages and distributed unimodally, ranging approximately from 200-1500 microns 2 with the largest somata occurring in the older age group. The range of microvessel diameters was also comparable but the largest microvessels were seen in the older animals. The histological arrangement of the ganglion permitted analyses to be made of 'neuronal' and 'axonal' areas independently. The number of microvessels per unit area was less in regions of the ganglion occupied by axons at both ages. Random transects indicated that the percentage area occupied by neuron somata decreases and that of axons increases with age. Overall, however, the results suggest that the histological organization, the size of vagal sensory neurons, the ganglionic microvessels, and the relationship between them, does not change greatly in Wistar rats of up to 2 years of age. Ultrastructural features of the aged sensory neurons included the presence of secondary lysosomes, disrupted rough endoplasmic reticulum, swollen Golgi cisternae, and the presence of much filamentous material in the perikaryon similar to that seen in chromatolytic neurons. However, analysis of electron micrographs did not reveal significant changes in the numbers of mitochondria or Golgi bodies. There was an overall increased thickness in the microvascular wall in the older animals with the endothelium and pericyte covering being significantly increased, but the thickness of the basal lamina was unchanged. The activities of neuronal NADH tetrazolium reductase, succinate dehydrogenase and cytochrome oxidase were all increased with age. The results suggest that vagal sensory neurons are not greatly affected by age in the rat.

Age-related changes in the morphology of preganglionic neurons projecting to the rat hypogastric ganglion.

The aim of this study was to investigate age-related changes in preganglionic neurons of the lumbar and sacral spinal cord of the male rat that may underlie impaired control of the urogenital system in old age. Preganglionic sympathetic and parasympathetic neurons of 4- and 24-month-old rats were identified by retrograde axonal tracing with cholera toxin subunit-B followed by immunocytochemistry. Labelled preganglionic neurons were scanned on 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 dendritic branch points and total dendritic length of sympathetic preganglionic neurons in the aged rats compared to the adult group. The soma area and number of primary dendrites were not significantly different. Some cells exhibited signs of degeneration, such as swelling of the soma and distension of the proximal part of primary dendrites. No significant differences were found in any of the parameters measured for the parasympathetic neurons. The changes in dendritic morphology of sympathetic preganglionic neurons may reflect altered central and peripheral control of pelvic viscera in old age.