Biomedical subjects

Janet R Keast

Publications and source records attributed to Janet R Keast.

9 recordsLinked to original sources

PubMed2006

Structural effects and potential changes in growth factor signalling in penis-projecting autonomic neurons after axotomy.

BACKGROUND: The responses of adult parasympathetic ganglion neurons to injury and the neurotrophic mechanisms underlying their axonal regeneration are poorly understood. This is especially relevant to penis-projecting parasympathetic neurons, which are vulnerable to injury during pelvic surgery such as prostatectomy. We investigated the changes in pelvic ganglia of adult male rats in the first week after unilateral cavernous (penile) nerve axotomy (cut or crush lesions). In some experiments FluoroGold was injected into the penis seven days prior to injury to allow later identification of penis-projecting neurons. Neurturin and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors for penile parasympathetic neurons, so we also examined expression of relevant receptors, GFRalpha1 and GFRalpha2, in injured pelvic ganglion neurons. RESULTS: Axotomy caused prolific growth of axon collaterals (sprouting) in pelvic ganglia ipsilateral to the injury. These collaterals were most prevalent in the region near the exit of the penile nerve. This region contained the majority of FluoroGold-labelled neurons. Many sprouting fibres formed close associations with sympathetic and parasympathetic pelvic neurons, including many FluoroGold neurons. However immunoreactivity for synaptic proteins could not be demonstrated in these collaterals. Preganglionic terminals showed a marked loss of synaptic proteins, suggesting a retrograde effect of the injury beyond the injured neurons. GFRalpha2 immunofluorescence intensity was decreased in the cytoplasm of parasympathetic neurons, but GFRalpha1 immunofluorescence was unaffected in these neurons. CONCLUSION: These studies show that there are profound changes within the pelvic ganglion after penile nerve injury. Sprouting of injured postganglionic axons occurs concurrently with structural or chemical changes in preganglionic terminals. New growth of postganglionic axon collaterals within the ganglion raises the possibility of the formation of aberrant synaptic connections between injured and un-injured ganglion neurons. Together these changes demonstrate a broader effect on the pelvic autonomic circuitry than simply loss of neuroeffector connections. These structural changes are accompanied by potential changes in neurotrophic factor signalling due to altered expression of receptors for members of the GDNF family. Together our results advance understanding of the responses of pelvic autonomic nerve circuits to injury and may assist in designing strategies for promoting regeneration.

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PubMed2006

Loss of nitrergic neurotransmission to mouse corpus cavernosum in the absence of neurturin is accompanied by increased response to acetylcholine.

The neurotrophic factor, neurturin (NTN), plays an important role in parasympathetic neural development. In the penis, parasympathetic nitrergic/cholinergic nerves mediate the erectile response. However, despite reduced parasympathetic penile innervation in mice lacking the NTN receptor, glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)2, they are capable of erection and reproduction. Our aim was to assess neural regulation of erectile tissues from mice lacking NTN. Responses of cavernosal smooth muscle were studied in vitro, monitoring agonist- and nerve-evoked changes in tension. Frequency-dependent nerve-evoked relaxations in the presence of guanethidine were markedly reduced in the mutant mice compared to wild types (19 vs 72% of phenylephrine pre-contraction). Atropine reduced the amplitude in wild-type mice to 61%, but abolished relaxations in knockout mice. In wild-type and knockout animals, nitric oxide synthase inhibition abolished neurogenic relaxations. In NTN knockout animals, EC(50) values for nitric oxide-dependent relaxations to acetylcholine and muscarine were increased approximately 0.5 log units. In contrast, contractions to electrical stimulation or phenylephrine, and relaxations to bradykinin or the nitric oxide donor, sodium nitroprusside, were unaltered. Immunohistochemistry confirmed that nerves immunoreactive for nitric oxide synthase, vesicular acetylcholine transporter and vasoactive intestinal polypeptide were substantially reduced in cavernosum of NTN knockout mice. Parallel immunohistochemical and pharmacological studies in GFRalpha2 knockout animals showed the same changes from their wild types as the NTN knockout animals. The data demonstrate that NTN is essential for normal development of penile erection-inducing nerves and that its absence leads to increased responsiveness to muscarinic agonists, possibly as a compensatory mechanism.

Acetylcholine↗

PubMed2006

Plasticity of pelvic autonomic ganglia and urogenital innervation.

Pelvic ganglia contain a mixture of sympathetic and parasympathetic neurons and provide most of the motor innervation of the urogenital organs. They show a remarkable sensitivity to androgens and estrogens, which impacts on their development into sexually dimorphic structures and provide an array of mechanisms by which plasticity of these neurons can occur during puberty and adulthood. The structure of pelvic ganglia varies widely among species, ranging from rodents, which have a pair of large ganglia, to humans, in whom pelvic ganglion neurons are distributed in a large, complex plexus. This plexus is frequently injured during pelvic surgical procedures, yet strategies for its repair have yet to be developed. Advances in this area will come from a better understanding of the effects of injury on the cellular signaling process in pelvic neurons and also the role of neurotrophic factors during development, maintenance, and repair of these axons.

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PubMed2006

Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons.

Spinal cord injuries often lead to disorders in the control of autonomic function, including problems with blood pressure regulation, voiding, defecation and reproduction. The root cause of all these problems is the destruction of brain pathways that control spinal autonomic neurons lying caudal to the lesion. Changes induced by spinal cord injuries have been most extensively studied in sympathetic preganglionic neurons, cholinergic autonomic neurons with cell bodies in the lateral horn of thoracic and upper lumbar spinal cord that are the sources of sympathetic outflow. After an injury, sympathetic preganglionic neurons in mid-thoracic cord show plastic changes in their morphology. There is also extensive loss of synaptic input from the brain, leaving these neurons profoundly denervated in the acute phase of injury. Our recent studies on sympathetic preganglionic neurons in lower thoracic and upper lumbar cord that regulate the pelvic viscera suggest that these neurons are not so severely affected by spinal cord injury. Spinal interneurons appear to contribute most of the synaptic input to these neurons so that injury does not result in extensive denervation. Since intraspinal circuitry remains intact after injury, drug treatments targeting these neurons should help to normalize sympathetically mediated pelvic visceral reflexes. Furthermore, sympathetic pelvic visceral control may be more easily restored after an injury because it is less dependent on the re-establishment of direct synaptic input from regrowing brain axons.

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PubMed2005

Enkephalin-immunoreactive interneurons extensively innervate sympathetic preganglionic neurons regulating the pelvic viscera.

Enkephalin (ENK)-immunoreactive (IR) axons occur in regions containing spinal autonomic neurons and endogenous opiates contribute to spinal regulation of bladder function. To identify possible spinal sites of opiate action, we used immunocytochemistry for ENK with retrograde tracing from the major pelvic ganglion (MPG), a key location for postganglionic neurons controlling pelvic viscera, with cholera toxin B subunit (CTB) or CTB-horseradish peroxidase (CTB-HRP). We compared the relationship of ENK-IR axons with sympathetic preganglionic neurons (SPNs) projecting to the MPG between intact spinal cords and cords with 2- or 11-week complete transections between thoracic segments 4 and 5. By light microscopy, sections of intact cord showed dense networks of ENK-IR axons surrounding CTB-IR SPNs in the intermediolateral cell column (IML), intercalated nucleus, and central autonomic area of lower thoracic and upper lumbar cord. This staining pattern was similar in rats with 2- or 11-week transections. Ultrastructurally, ENK-IR axons formed synapses on SPNs in all three autonomic subnuclei of intact cord. In the IML, ENK-IR varicosities contributed 52% of the synapses on the somata of MPG-projecting SPNs. In 2-week transected cord, synapses from ENK-IR axons persisted on SPNs and the proportion of input to IML SPNs had increased to 67%, probably reflecting loss of supraspinal input. These results suggest that endogenous opioids could play a major role in controlling sympathetic outflow to the bladder through a direct action on SPNs. The persistence of the dense ENK innervation after complete cord transection indicates that the ENK-IR input to SPNs arises predominantly from intraspinal sources.

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PubMed2005

Neurturin has multiple neurotrophic effects on adult rat sacral parasympathetic ganglion neurons.

Neurturin (NTN) is an important neurotrophic factor for parasympathetic neurons; however, no studies to date have investigated the signalling mechanisms downstream of GFRalpha2 and Ret activation underlying this neurotrophic support. This is particularly important for pelvic parasympathetic neurons, which are prone to injury during surgical procedures such as prostatectomy, and where there are no current therapies for axonal regeneration. To address this issue we have cultured dissociated adult rat pelvic ganglion neurons and also examined the structural changes in pelvic ganglion neurons after axotomy. Axotomised penile neurons deprived of target-derived support had smaller somata than intact neurons. Studies of cultured adult pelvic ganglion neurons also demonstrated that NTN stimulated soma growth. Further experiments showed that NTN reduced the up-regulation of tyrosine hydroxylase expression in cultured pelvic parasympathetic neurons. NTN stimulated the extension of neurites in cultured parasympathetic, but not sympathetic, pelvic ganglion neurons. Inhibition of phosphatidylinositol 3-kinase prevented initiation of neurite outgrowth, whereas inhibition of the mitogen-activated protein kinase and the Src family kinase pathways disrupted NTN-stimulated microtubule assembly. Surprisingly, NTN did not activate the transcription factor cAMP-response element binding protein (CREB), which is typically involved in neurotrophic signalling in sympathetic neurons. This is the first study to identify signalling pathways activated by NTN in adult parasympathetic neurons. Our results may lead to a better understanding of regenerative mechanisms in parasympathetic neurons, especially for those innervating urogenital organs. Our results also indicate that neurotrophic signalling in parasympathetic neurons is different from that in other types of peripheral neurons.

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PubMed2004

Artificial autonomic reflexes: using functional electrical stimulation to mimic bladder reflexes after injury or disease.

Autonomic reflexes controlling bladder storage (continence) and emptying (micturition) involve spinal and supraspinal nerve pathways, with complex mechanisms coordinating smooth muscle activity of the lower urinary tract with voluntary muscle activity of the external urethral sphincter (EUS). These reflexes can be severely disrupted by various diseases and by neurotrauma, particularly spinal cord injury (SCI). Functional electrical stimulation (FES) refers to a group of techniques that involve application of low levels of electrical current to artificially induce or modify nerve activation or muscle contraction, in order to restore function, improve health or rectify physiological dysfunction. Various types of FES have been developed specifically for improving bladder function and while successful for many urological patients, still require substantial refinement for use after spinal cord injury. Improved knowledge of the neural circuitry and physiology of human bladder reflexes, and the mechanisms by which various types of FES alter spinal outflow, is urgently required. Following spinal cord injury, physical and chemical changes occur within peripheral, spinal and supraspinal components of bladder reflex circuitry. Better understanding of this plasticity may determine the most suitable methods of FES at particular times after injury, or may lead to new FES approaches that exploit this remodeling or perhaps even influence the plasticity. Advances in studies of the neuroanatomy, neurophysiology and plasticity of lumbosacral nerve circuits will provide many further opportunities to improve FES approaches, and will provide "artificial autonomic reflexes" that much more closely resemble the original, healthy neuronal regulatory mechanisms.

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PubMed2003

Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder.

We have investigated whether bladder afferent neurons are likely to be targets for circulating estrogens by mapping estrogen receptor (ER) distribution in lumbosacral dorsal root ganglia (DRG) of adult female rats. Sensory neurons innervating either the detrusor or trigone regions were identified by application of fluorescent retrograde tracer dyes to the bladder wall. Labelled neurons were classified by their immunoreactivity for either type of ER (ERalpha or ERbeta) and further compared with subpopulations of neurons containing substance P, calcitonin gene-related peptide and vanilloid receptor (a marker of polymodal nociceptors). Both ER types were expressed in numerous sensory neurons of either upper lumbar (L1/L2) or lower lumbar/sacral (L6/S1) ganglia and there was almost complete coexpression of ERalpha and ERbeta. ER-positive neurons were mainly small-medium size (18-25-microm diameter), indicating that they may be nociceptors and/or supply visceral targets. Most bladder-projecting neurons expressed ERs and the majority of these also expressed neuropeptides or vanilloid receptor. Afferent neurons supplying detrusor and trigone regions had similar immunohistochemical features. About a third of the bladder-projecting neurons expressed both ER and vanilloid receptor, suggesting a mechanism by which estrogens could influence bladder pain. The prevalence of different chemical classes of ER-positive bladder-projecting neurons was reflected throughout the entire population of neurons in dorsal root ganglia of these spinal levels, suggesting that neurons supplying other pelvic visceral targets may have similar chemical profiles. These results suggest that many functional classes of sensory neurons innervating the lower urinary tract are likely to be targets for circulating estrogens, including many nociceptor neurons. The coexistence of ERalpha and ERbeta suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system.

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PubMed2003

Immunohistochemical characterisation of pelvic autonomic ganglia in male mice.

Pelvic ganglia are mixed sympathetic-parasympathetic ganglia and provide the majority of the autonomic innervation to the urogenital organs. Here we describe the structural and histochemical features of the major pelvic ganglion in the male mouse and compare two different mouse strains. The basic structural features of the ganglion are similar to those in the male rat. Almost all pelvic ganglion cells are monopolar and most are cholinergic. All contain either neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP), or both peptides together. The peptide coexistence varies between strains, with C57BL/6 mice having similar proportions of neurons with NPY alone, VIP alone or both peptides. In contrast, virtually all pelvic neurons in the Quackenbush-Swiss (QS) strain express NPY, i.e. the level of VIP/NPY coexistence is much higher. Cholinergic axons provide the major nerve supply to epithelia of reproductive organs, bladder smooth muscle and, as described previously, penile erectile tissue. They also provide a minor component of the smooth muscle innervation of the prostate gland, seminal vesicles and vas deferens. Virtually all non-cholinergic pelvic ganglion cells are noradrenergic and contain NPY. Their major target is smooth muscle of reproductive organs. This study shows that the male mouse pelvic ganglion bears many similarities to that in the rat, but that VIP/NPY colocalisation is much more common in the mouse. We also show that there are differences in peptide expression in parasympathetic pelvic neurons between strains of mice. These studies provide the framework for future investigations on neural regulation of urogenital function, particularly in transgenic and knockout models.

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