Upregulation of epidermal gap junctional proteins in patients with venous disease.

BACKGROUND: Leg ulceration is a feared complication of venous insufficiency. It is not known whether varicose veins predispose skin to poor wound healing. The expression pattern of gap junctional protein connexin, a known marker of poor wound healing, was investigated across various stages of venous disease. METHODS: Patients undergoing intervention for varicose veins were assessed according to the Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification of varicose veins. Paired 4-mm punch biopsies were taken from above the ankle (pathological) and above the knee (control). Tissues were stained with haematoxylin and eosin, and for connexin 43, connexin 30 and connexin 26. RESULTS: Forty-eight paired biopsies were taken (12 each for CEAP class C0, C2, C4 and C6). The pathological skin showed progressive epithelial hyperthickening, an increase in the number and depth of rete ridges, increased inflammation and loss of dermal architecture with disease progression from C4 onwards. The overall absolute connexin expression and mean connexin expression per cell in the pathological skin similarly increased across the CEAP classes from as early as C2. Increasing levels of connexin in control skin were also noted, indicating progression of the disease proximally. Connexin 43 expression showed the strongest positive correlation between pathological and control skin. CONCLUSION: Connexins were overexpressed in patients with simple varicose veins, with a stepwise increased expression through venous eczema to ulceration. Connexin 43 is a potential biomarker for venous disease. This finding suggests that varicose veins predispose skin to poor wound healing. Surgical relevance The overexpression of connexins, a family of gap junctional proteins, is known to cause poor healing in venous leg ulceration. It is not known whether there is any association with superficial venous disease. Here, connexin proteins were overexpressed in patients with uncomplicated varicose veins, before histological skin changes. Connexin could be a biomarker of venous disease progression.

Abnormal connexin expression in human chronic wounds.

BACKGROUND: Regulated alteration of connexin expression has been shown to be integral to acute wound repair. Downregulation of the gap-junction protein connexin 43 at the wound edge has been correlated with keratinocyte and fibroblast migration, while abnormal overexpression of connexin 43 significantly perturbs healing, as shown in the streptozotocin diabetic rodent impaired healing model. OBJECTIVES: To examine the protein expression levels of connexin 43, in addition to connexins 26 and 30, in a variety of human chronic wounds. METHODS: Wound-edge punch biopsies and a matched control from the arm were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared with that in a matched, nonwounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification. RESULTS: Epidermal connexin 43, connexin 26 and connexin 30, and dermal connexin 43 were discovered to be strikingly upregulated in every ulcer from all three wound types, pointing to connexin upregulation as a common feature between chronic wounds. CONCLUSIONS: This result supports efforts to target connexin 43 to promote cell migration and wound healing in chronic ulcers.

Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane.

INTRODUCTION: There is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E2 (PGE2) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain. METHODS: Human amniotic membranes isolated from the cervix (CAM) or placenta regions (PAM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE2, collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay. RESULTS: Values for tensile strength were significantly higher in PAM than CAM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE2 release and GAG levels in PAM and CAM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in PAM and CAM. DISCUSSION: Fibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE2 release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content. CONCLUSION: A combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition.

Optical delineation of human malignant melanoma using second harmonic imaging of collagen.

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons.

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.

Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity.

OBJECTIVES: To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. METHODS: Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. RESULTS: Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. CONCLUSIONS: Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat.

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness.

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.

Serum-free culture of dissociated, purified adult and aged sympathetic neurons and quantitative assays of growth and survival.

In vitro studies of dissociated neurons have provided crucial data regarding the regulation of plasticity in embryonic and perinatal neurons from both central and peripheral nervous systems. There have been few attempts to apply these methods to adult or aged neurons and the methods that have been reported have not been able to dissect the possible confounding contributions of non-neuronal cells and serum. Furthermore, quantitative assays of cultured neurons, particularly of their growth, have rarely been described. We report here the development of a novel method for the dissociation, purification and culture of sympathetic neurons from the adult and aged rat SCG under serum free conditions and in defined media. The technique results in a relatively high yield of viable, growing neurons. We describe methods for assaying the total yield of neurons, the proportion of surviving neurons and the proportion of neurons initiating neurite outgrowth after plating. A novel semiautomated assay of neurite outgrowth is outlined using image analysis of composite images of immunofluorescence-stained single neurons.

Cavernous sinus ganglia are sources for parasympathetic innervation of cerebral arteries in rat.

Retrograde tracing and immunohistochemistry was used in rats to investigate whether the ganglia in the cavernous sinus contribute to cerebrovascular innervation. The cavernous sinus ganglia in rat include the cavernous part of the pterygopalatine ganglion (PGC) and small cavernous ganglia (CG). The tracers, fluorogold and fast blue, were applied to the middle cerebral artery in eight rats. After 1 to 4 days, the cavernous sinuses were dissected out and studied as whole mount preparations and sections. A moderate number of labeled neurons were visible in the ipsilateral PGC and CG. Furthermore, fibers in the cavernous nerve plexus and abducens nerve were labeled, suggesting that the pathway from the cavernous sinus ganglia to the cerebral arteries runs through the cavernous plexus and then retrogradely along the abducens nerve to the internal carotid artery. Selected sections were immunohistochemically stained for the cholinergic marker, vesicular acetylcholine transporter (VAChT). Most cells in the PGC and CG were VAChT-immunoreactive, some of which also contained tracer. It is concluded that in rat, the cavernous sinus ganglia, consisting of the PGC and small CG, contribute to parasympathetic cerebrovascular innervation and that the cavernous nerve plexus and abducens nerve are involved in the pathway from these ganglia to the cerebral arteries.

NGF expression in the aged rat pineal gland does not correlate with loss of sympathetic axonal branches and varicosities.

The factors that determine the ability of some, but not all neurons, to sustain their axonal projections during aging remain largely unknown. Because sympathetic neurons remain responsive to nerve growth factor (NGF) in old age, it has been proposed that the selective decrease observed in the sympathetic innervation to some targets in aged rats may be the result of a deficit in target-derived NGF. In this study we utilized two different techniques to demonstrate decreased target innervation by sympathetic fibers in the aged rat pineal gland, which is an appropriate and relevant model for examining mechanisms of neuron-target interactions in aging. Tyrosine hydroxylase immunoreactive profiles were quantified in pineal glands of young and aged male Sprague-Dawley rats. The density of tyrosine hydroxylase-immunoreactive fibers was 30% lower in aged pineals, although the remaining fibers contained 20% more tyrosine hydroxylase-immunoreactivity. Othograde tracing of the pineal sympathetic innervation using biotinylated dextran revealed that average axon length, varicosity numbers, branch point numbers, and numbers of terminations were all decreased by approximately 50% in aged tissues, indicating possible functional deficits. These findings suggest that whole branches, along with their associated varicosities were lost in old age. A sensitive quantitative ribonuclease protection assay and a two-site ELISA assay were used to examine whether reduced NGF availability might correlate with sympathetic nerve atrophy. No significant differences were detected in either NGF mRNA or NGF protein levels when comparing young and aged pineal glands, suggesting that atrophy in aged sympathetic neurons is not causally related to reduced availability of NGF at the target. Our results indicate that mechanisms other than NGF expression need to be explored in order to explain the age-related axonal regression observed in this target.

In oculo transplants of myometrium from postpartum guinea pigs fail to support sympathetic reinnervation.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

Imaging and analysis of perivascular nerves in human mesenteric and coronary arteries: a comparison between epi-fluorescence and confocal microscopy.

Perivascular nerves supplying human arteries can be visualised after immunohistochemical staining for a variety of markers. The pattern and density of perivascular nerves vary with region, age and disease. Quantification of the nerve plexus, which may be performed by image analysis, is a prerequisite to assess differences in nerve density. The use of epi-fluorescence microscopy (EFM) presents difficulties in visualising the nerve plexus in certain tissues, which can affect the reliability with which specific staining can be localised and distinguished from non-specific staining. In this study, confocal scanning laser microscopy (CSLM) was used in parallel with EFM, in order to compare images from both techniques. In a comparison of identical areas of nerve plexuses of human mesenteric and coronary arteries stained for protein gene product (PGP) 9.5 and imaged using CSLM and EFM, higher values for area percent (area occupied by nerves), and intercept density (ID/mm, which reflects the number of nerve bundles detected) were found in CSLM images. Similar comparisons of unmatched epi-fluorescence and confocal images from a group of 45 mesenteric arteries revealed no significant difference for area percent, but significantly higher values for ID/mm in CSLM images. These findings illustrate that the better image quality in CSLM influences image analysis and can be very useful in studies of dynamic changes in nerve plexuses. We recommend CSLM for tissues that suffer from high background staining, such as human mesenteric and coronary arteries.

Gonadotropin-releasing hormone immunoreactivity in the nasal epithelia of adults with Kallmann's syndrome and isolated hypogonadotropic hypogonadism and in the early midtrimester human fetus.

GnRH-secreting neurons are known to originate in the epithelium of the medial olfactory placode, whence they migrate along the axons of the terminal nerve via the forebrain and into the hypothalamus. Synaptic contact between the developing olfactory bulbs and fascicles of the vomeronasal, terminal, and olfactory nerves does not occur in Kallmann's syndrome. Consequently, there is migration arrest of GnRH cells and partial or complete failure of formation of the olfactory bulbs, resulting in severe olfactory deficit and hypogonadotropic hypogonadism. In the present study, using an immunofluorescent, double immunostaining technique and confocal laser scanning microscopy, we observed GnRH-immunoreactive neurons in the hypothalamus of a 14-week-old human fetus. However, migration of GnRH neurons was not complete, and indeed, such cells were seen to be migrating along terminal nerve fascicles beneath the cribriform plate in a 16-week-old fetus. The same immunofluorescent technique demonstrated the presence of GnRH cells in biopsies of nasal mucosa obtained from three adults with Kallmann's syndrome, one normosmic subject with hypogonadotropic hypogonadism, and a eugonadal male cadaver. These findings are consistent with two different interpretations: the nasal GnRH neurons may be vestigial, representing cells that failed to migrate during embryogenesis; alternatively, they may have been generated de novo later in life, a possibility consistent with the recognized plasticity of human postnatal olfactory neuroepithelium. They also reveal that subjects with the normosmic (i.e. non-Kallmann's) form of GnRH deficiency are able to synthesize immunologically recognizable GnRH, implying that failure of GnRH synthesis is not responsible for this type of hypogonadotropic hypogonadism.

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH)). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Target-specific differences in the dendritic morphology and neuropeptide content of neurons in the rat SCG during development and aging.

Our purpose in this work was to investigate the role of target tissues in the regulation of dendritic morphology from sympathetic neurons during development and aging. Neurons were retrogradely labeled from three targets, the iris, the submandibular gland (SMG), and the middle cerebral artery (MCA). They were then fixed and intracellularly injected to demonstrate their dendritic arborizations. Dendritic geometry varied quantitatively in sympathetic neurons innervating different target tissues at all stages of development. Neurons innervating the iris had the largest cell bodies and most extensive dendritic arborizations, whereas the vasomotor neurons were the smallest. The number of primary dendrites, however, did not vary significantly between the different neuronal populations. The growth of dendritic arborizations during postnatal development and their atrophy in old age were not concordant in the different neuron populations we studied. Neurons innervating the MCA and the iris ceased dendritic growth early in postnatal development, whereas the dendritic complexity of neurons supplying the SMG increased well into adulthood. By contrast, dendritic atrophy was seen in aged MCA- and SMG-projecting neurons but not in those innervating the iris, suggesting, with other evidence, correlated and distinct patterns of growth and atrophy in axons and dendrites of mature sympathetic neurons projecting to different targets. Swollen dendrites and protuberances on cell soma were a prominent feature of aged neurons. In addition to the target-specific variation in neuronal morphology, we observed diversity in neurotransmitter phenotype. For example, neuropeptide Y was expressed in iridial but not SMG-projecting neurons. These results show a range of age- and target-specific differences in the dendritic morphology and neuropeptide content of sympathetic neurons that may be a result of differing trophic interactions with their target tissues.

Pre-ulcerative villous contraction and microvascular occlusion induced by indomethacin in the rat jejunum: a detailed morphological study.

BACKGROUND: In indomethacin-induced jejunal ulceration in the rat, villi undergo both early microvascular injury and shortening that may involve activation of villous smooth muscle. AIM: This study sought to substantiate light microscopic observations using three-dimensional imaging of early villous architectural changes in response to indomethacin. METHODS: At both 2 and 6 h after oral indomethacin 15 mg/kg or vehicle to groups of rats, the vasculature of the small intestine was visualised by both carbon-ink perfusion/confocal microscopy and injection casting. The mucosa was also examined for lesions by dissection microscopy and scanning electron microscopy. RESULTS: In indomethacin-dosed rats examined by scanning electron microscopy and histology, the mucosa at 2 h showed villous shortening and wrinkling of the surface epithelium without epithelial loss; at 6 h, the mucosa was flattened, often with epithelial loss to expose a 'contracted' villous core. Examination of the 'vasculature in carbon-injected tissues indicated significant reductions of both mucosal height and inter-capillary distance at both 2 and 6 h post-indomethacin. Scanning electron microscopy of injection casts at 2 and 6 h indicated similar changes. These changes were not seen in control tissues. CONCLUSION: Histology, confocal microscopy and scanning electron microscopy support the proposal that villous shortening with disruption of surface capillary architecture are early changes in the ulcerative enteropathy induced by nonsteroidal anti-inflammatory drugs.

Regulation of rat sympathetic nerve density by target tissues and NGF in maturity and old age.

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of approximately 50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density approximately 50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target-derived neurotrophic factors.