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ABSTRACT: To evaluate whether augmenting neuronal protective mechanisms might slow or arrest experimental diabetic peripheral neuropathy (DPN). DPN is one of the most common neurodegenerative disorders and is rising in prevalence. How it targets sensory neurons is uncertain; the disorder is irreversible and untreatable. We explored the intrinsic protective properties of overexpressed human HSP27 on experimental DPN. HSP27 is a small pro-survival heat shock protein that also increases axonal regeneration.
Experimental diabetes was superimposed on mice overexpressing a human HSP27 transgene and its impact was evaluated on epidermal innervation, behavioral tests of sensation and electrophysiological indices of DPN.
Mice that overexpress human HSP27 in their sensory and motor neurons and that were made diabetic for 6 months by streptozotocin treatment were protected from a range of neuropathic abnormalities, including loss of footpad thermal sensation, mechanical allodynia, loss of epidermal innervation, and slowing of sensory conduction velocity. The protection was selective for sensory neurons in comparison to motor neurons and at 6 months provided better protection in female than male mice. Markers of RAGE-NFκB activation were attenuated by the transgene.
The findings support the idea that diabetic polyneuropathy involves a unique, sensory-centric neurodegenerative process which can be reduced by overexpressing a single gene, an important starting point for new disease-modifying therapeutic approaches.
Neurobiology of Disease 05/2012; 47(3):436-43. · 5.40 Impact Factor
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ABSTRACT: We identify an essential role for the RGD (Arg-Gly-Asp tripeptide) moiety in vivo during adult peripheral neuron regenerative growth. Beyond a peripheral nerve transection there were rises in the fibronectin extracellular matrix, and striking rises in the mRNA and protein expression of integrin subunits sensitive to RGD/fibronectin signalling. Neuron perikarya, axons and Schwann cells all expressed RGD/fibronectin sensitive integrins after injury. To evaluate the significance of RGD/fibronectin-integrin interactions, we infused solutions of a pentapeptide including the RGD motif (sRGD) serially and directly within the milieu of early axon growth across rat sciatic transection injuries. While low dose infusions of sRGD facilitated early axon ingrowth, we encountered inhibition of ingrowth and bridge formation with higher doses of sRGD indicating competitive disruption of RGD/fibronectin-integrin signalling. Fibronectin RGD moieties serve a critical and important role during peripheral axon outgrowth.
Neurobiology of Disease 01/2009; 34(1):11-22. · 5.40 Impact Factor
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ABSTRACT: Diabetes mellitus targets the peripheral nervous system in unique but disabling ways. Although several mechanisms may target peripheral neurons, they render a degenerative pattern of damage that begins in distal terminals. Moreover, sensory neurons are involved early, motor neurons later. By studying a variety of diabetic neuropathy models in rats, mice, and other species, an overall appreciation of its neurodegeneration emerges. Understanding how mechanisms of diabetes complications target peripheral neurons selectively may offer opportunities to intervene before irretrievable neuron loss develops.
The Neuroscientist 08/2008; 14(4):311-8. · 4.57 Impact Factor
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ABSTRACT: Erythropoietin (EPO) and its receptor (EPO-R), mediate neuroprotection from axonopathy and apoptosis in the peripheral nervous system (PNS). We examined the impact and potential mechanisms of local EPO signaling on regenerating PNS axons in vivo and in vitro. As a consequence of injury, peripheral nerve axons and DRG neurons have a marked increase in the expression of EPO and EPO-R. Local delivery of EPO via conduit over 2 weeks to rat sciatic nerve following crush injury increased the density and maturity of regenerating myelinated axons growing distally from the crush site. In addition, EPO also rescued retrograde degeneration and atrophy of axons. EPO substantially increased the density and intensity of calcitonin gene-related peptide (CGRP) expression within outgrowing axons. Behavioral improvements in sensorimotor function also occurred in rats exposed to near nerve EPO delivery. EPO delivery led to decreased nuclear factor kappaB (NFkB) activation but increased phosphorylation of Akt and STAT3 within nerve and dorsal root ganglia neurons indicating rescue from an injury phenotype. Spinal cord explant studies also demonstrated a similar dose-dependent effect of EPO upon motor axonal outgrowth. Local EPO signaling enhances regenerating peripheral nervous system axons in addition to its known neuroprotection. Exogenous EPO may have a therapeutic role in a large number of peripheral nerve diseases through its impact on regeneration.
Neuroscience 07/2008; 154(2):767-83. · 3.38 Impact Factor
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ABSTRACT: During regeneration of injured peripheral nerves, local conditions may influence how regenerative axon sprouts emerge from parent axons. More extensive lesions might be expected to disrupt such growth. In this work, we discovered instead that long segmental crush injuries facilitate the growth and maturation of substantially more axon sprouts than do classical short crush injuries (20 mm length vs. 2 mm). At identical distances from the proximal site of axon interruption there was a 45% rise in the numbers of neurofilament labeled axons extending through a long segmental crush zone by 1 week. By 2 weeks, there was a 35% greater density of regenerating myelinated axons in long compared with short crush injuries just beyond (5 mm) the proximal injury site. Moreover, despite the larger numbers of axons, their maturity was identical and they were regular, parallel, associated with Schwann cells (SCs) and essentially indistinguishable between the injuries. Backlabeling with Fluorogold indicated that despite these differences, the axons arose from similar numbers of parent motor and sensory neurons. Neither injury was associated with ischemia. Both injuries were associated with rises in GFAP (glial acidic fibrillary protein) and p75 mRNAs, markers of SC plasticity but p75, GFAP and brain-derived neurotrophic factor mRNAs did not differ between the injuries. There was a higher local mRNA level of GAP43/B50 at 7 days following injury and a higher sonic hedgehog protein (Shh) mRNA at 24 h in long crush zones. GAP43/B50 protein and SHH protein both had prominent localization within regenerating axons. Long segmental nerve trunk crush injuries do not impair regeneration but instead generate greater axon plasticity that results in larger numbers of mature myelinated axons. The changes occur without apparent change in SC activation, overall nerve architecture or nerve blood flow. While the mechanism is uncertain, the findings indicate that manipulation of the nerve microenvironment can induce substantial changes in regenerative sprouting.
Neuroscience 05/2008; 152(4):877-87. · 3.38 Impact Factor
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ABSTRACT: The regeneration of adult peripheral neurons after transection is slow, incomplete and encumbered by severe barriers to proper regrowth. The role of RHOA GTPase has not been examined in this context. We examined the expression, activity and functional role of RHOA GTPase and its ROK effector, inhibitors of regeneration, during peripheral axon outgrowth. We used qRT-PCR, quantitative immunohistochemistry, and assays of RHOA activation to examine expression in sensory neurons of rats with sciatic transection injuries. In vitro, we exposed dissociated adult sensory neurons, not grown on inhibitory substrates, to a RHOA-ROK inhibitor HA-1077 and measured neurite initiation and outgrowth. In vivo, we exposed early regenerating axons and Schwann cells directly to HA-1077 in a conduit connecting the proximal and distal stumps of transected sciatic nerves. Intact adult dorsal root ganglia sensory neurons expressed RHOA and ROK 1 mRNAs and protein and there were rises in RHOA after injury. Activated GTP-bound RHOA, undetectable in intact ganglia, was dramatically upregulated in both neurons and axons after injury. Adult rat sensory neurons in vitro demonstrated a dose-related increase in the initiation of neurite outgrowth, and in the proportion with long neurites when they were exposed to a ROK antagonist. Regenerative bridges that were directly exposed to the ROK inhibitor had a dose-related rise in the extent and distance of in vivo axon and partnered Schwann cell regrowth within them. RHOA activation and signaling are features of adult peripheral axon regeneration within its own milieu, independent of myelin. Inhibition of its activation may benefit peripheral axon lesions.
Experimental Neurology 05/2008; · 4.70 Impact Factor
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ABSTRACT: To examine the epidemiology of Guillain-Barré syndrome (GBS) in Alberta between 1994 and 2004 with data derived from hospital administration procedures.
Data from 3,959,857 individuals (1,956,841 females and 2,003,016 males) aged 1-110 years and residing in Alberta, Canada, were included in the analysis. A Poisson regression analysis was performed to determine the predictors of GBS events.
After age and sex standardisation to the 2001 Canadian census population, incidence rates ranged from 0.97 to 2.32 per 100,000 over the course of the 11-year period, with a mean incidence of 1.6 per 100,000. Significant effects of gender, age group and year were found. Males were found to be 1.5 times more likely to acquire GBS than females. Relative to those in their first decade, the risk of acquiring GBS increased with advancing age, whereby the incidence in males peaked in the 7th decade of life and in females in the 8th decade of life. The incidence fluctuated over the 11-year period, with a minimum in 1998 and a maximum in 2004.
The incidence of GBS in Alberta between 1994 and 2004 fluctuated within a narrow range, was similar to that previously reported worldwide, demonstrated a male preponderance and increased in elderly patients.
Journal of neurology, neurosurgery, and psychiatry 04/2008; 79(3):318-20. · 4.87 Impact Factor
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Cephalalgia 09/2006; 26(8):1028-30. · 3.43 Impact Factor
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ABSTRACT: Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both alpha and beta insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor alpha subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.
Neuroscience 06/2006; 139(2):429-49. · 3.38 Impact Factor
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ABSTRACT: The support of distal regenerating axons and epidermal nerve fibres through growth factor delivery may depend on the site of delivery. While low-dose systemic insulin provides trophic support for regenerating axons or axons from diabetic animals, its potential action upon the most distal neurites within the epidermis is unknown. In diabetic neuropathy, distal loss of axons is an important clinical and pathological feature. We hypothesised that insulin and IGF-1 delivered intrathecally could support the most distal epidermal nerve fibres.
As insulin and IGF-1 receptors are present upon sensory ganglion perikarya, we studied the impact of intrathecal delivery of low-dose insulin and equimolar IGF-1 on the density of epidermal axons expressing protein gene product 9.5 in experimental diabetic rats. After 2 months of diabetes induced by streptozotocin injection, intrathecal delivery of low-dose insulin or IGF-1 or saline was provided for 1 month, with comparison to compatible doses of subcutaneous insulin delivery.
Diabetes, in itself, was associated with a decline in epidermal nerve fibre density. Delivery of both intrathecal IGF-1 and insulin was associated with significant improvement in epidermal fibre density (greatest with IGF-1) and length relative to placebo.
Central intrathecal delivery of IGF-1 and insulin offers remote support for epidermal nerve fibres, subjected to 'dying-back' in early diabetic polyneuropathy.
Diabetologia 06/2006; 49(5):1081-8. · 6.81 Impact Factor
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ABSTRACT: Insulin peptide has been identified to promote regeneration of axons in culture and in some in vivo model systems. Such actions have been linked to direct actions of insulin, or to cross occupation of closely linked IGF-1 receptors. In this work, we examined insulin support of peripheral nerve regenerative events in mice. Systemic insulin administration accelerated the reinnervation of foot interosseous endplates by motor axons after sciatic nerve transection and enhanced recovery of functional mouse hindpaw function. Similarly, insulin accelerated the regeneration-related maturation of myelinated fibers regrowing beyond a sciatic nerve crush injury. That such benefits might occur through direct signaling on axons was supported by immunohistochemical studies of expression with an antibody directed to the beta insulin receptor (IR) subunit. The proportion of sensory neurons expressing IRbeta increased ipsilateral to a similar sciatic crush injury in the L4 and L5 dorsal root ganglia. Insulin receptors, although widely expressed in axons, were also preferentially and intensely expressed on axons regrowing just beyond a peripheral nerve crush injury zone. The findings indicate that insulin imparts a substantial impact on regenerating peripheral nerve axons through upregulation of its expression following injury. Although the findings do not exclude insulin coactivating IGF-1 receptors during regeneration, its own receptors are present and available for action on injured nerves.
Experimental Neurology 08/2004; 188(1):43-51. · 4.70 Impact Factor
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ABSTRACT: Sensory neurons in diabetes may be primarily targeted by diabetes and their involvement may account for prominent sensory loss and pain in diabetic patients. Previous studies demonstrating evidence of excessive polyol flux, microangiopathy, and oxidative stress involving sensory axons and ganglia have been joined by more recent work demonstrating altered neuron phenotype, mitochondrial dysfunction, ion channel alterations, and abnormal growth factor signaling. As such, an interesting and unique panoply of molecular changes in primary sensory neurons has been identified in diabetic models. Insulin deficiency and subsequent changes in second messenger signaling may also play an important role in how sensory neurons respond to diabetes. Applying approaches to support sensory neurons in diabetes may be an important therapeutic direction in diabetic patients.
Journal of Neuropathology and Experimental Neurology 07/2004; 63(6):561-73. · 4.26 Impact Factor
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ABSTRACT: Vacuolar changes in primary sensory neurons have been described in the context of disease and injury. In this work we examine microvacuolation of rat dorsal root ganglion (primary sensory) neurons. This change, attributed to swelling of mitochondria, had a direct relationship with the duration of the post mortem interval prior to harvesting. Similar microvacuolation, but also the presence of subplasmalemmal scalloping was prominent in dorsal root ganglion samples from patients without known peripheral disease undergoing post mortem examination. Both types of vacuolar change differ from "signet ring" vacuolation linked to neuron injury or disease. We suggest that care is required in evaluating vacuolar changes in primary sensory neurons. Microvacuolation is probably not a feature of sensory neuron disease.
Journal of Neurocytology 06/2003; 32(4):393-8. · 1.94 Impact Factor
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ABSTRACT: Three patients are described who had severe and progressive diabetic lumbosacral plexopathy despite active immunosuppressive therapy. One patient developed the condition while immunosuppressed for a cardiac transplant and two others progressed while receiving intravenous gamma globulin. The cases raise questions about current unsupported practices of treatment for this condition. Robust clinical trial evidence is required before immunosuppression can be recommended.
Acta Neurologica Scandinavica 05/2003; 107(4):299-301. · 2.47 Impact Factor
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ABSTRACT: In some disorders of the peripheral nervous system, it is relevant to understand how sensory neurons respond to selective ganglion ischemia. Sensory dorsal root ganglia may be susceptible to ischemic damage and irretrievable neuron loss because of their metabolic requirements. In diabetes, heightened sensitivity to ischemia associated with elevated endothelin levels might render ganglia particularly vulnerable. In this work, we created a model of local sensory ganglion ischemia by generating intense local vasoconstriction from applied endothelin-1 (ET). In this model, we compared relative vulnerability of L5 ganglia microvessels and neurons to ET in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic ganglia had reductions in baseline core ganglion blood flow (GBF) measured using microelectrode hydrogen clearance polarography and ET induced particularly profound declines. Serial GBF measurements made using a laser Doppler flowmetry probe also indicated that diabetic ganglia exposed to ET had a marked prolongation in its action. Neuron perikarya and proximal axon segments were more vulnerable in diabetes. Neurons exhibited loss of neurofilament labeling, dissolution of the neurons, replacement of neurons with "nests of Nageotte," displacement of nuclei to the periphery of perikarya, and nuclear labeling with TUNEL. Both intraganglionic axons and downstream sural sensory axons developed evidence of axonal degeneration. Local endothelin-induced vasoconstriction of microvessels supplying dorsal root ganglia provides a selective model of ischemia. Diabetic vessels and neurons, exposed to a greater depth and duration of ischemia from endothelin, are especially vulnerable.
Neuroscience 02/2003; 122(4):897-905. · 3.38 Impact Factor
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ABSTRACT: Modern surgical techniques cannot guarantee functional recovery following peripheral nerve injuries. Research into factors that may influence nerve regeneration has therefore assumed a prominent potential therapeutic role. We report here on the development of an approach to allow for direct manipulation of the microenvironment of regenerating peripheral nerve axons. We show that solutions can be delivered directly to this local milieu in vivo and that such a delivery can be performed multiple times over an extended period, potentially facilitating studies of multiple molecular players that act locally. We also demonstrate that the bundle of regenerated axons are amenable to morphological analysis by 21 days and that the injection system remains patent for at least 21 days.
Journal of Neuroscience Methods 02/2003; 122(2):171-8. · 1.98 Impact Factor
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Neurology 12/2002; 59(10):1660-1. · 8.31 Impact Factor
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ABSTRACT: Following injury to a peripheral nerve, changes in the behavior of Schwann cells help to define the subsequent microenvironment for regeneration. Such changes, however, have almost exclusively been considered in the context of Wallerian degeneration distal to an injury, where loss of axonal contact or input is thought to be critical to the changes that occur. This supposition, however, may be incorrect in the proximal stumps where axons are still in contact with their cell bodies. In this work, we studied aspects of in vivo Schwann cell behavior after injury within the microenvironment of proximal stumps of transected rat sciatic nerves, where axons are preserved. In particular we studied this microenvironment proximal to the outgrowth zone, in an area containing intact myelinated fibers and a perineurial layer, by using double immunolabelling of Schwann cell markers and 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells. In normal sciatic nerve, Schwann cells were differentiated, in an orderly fashion, into those associated with unmyelinated fibers that labeled with glial fibrillary acidic protein (GFAP) and those associated with myelinated fibers that could be identified by individual axons and myelin sheaths. After sciatic nerve transection, there was rapid and early expansion in the population of GFAP-labeled cells in proximal stumps that was generated in part, by de novo expression of GFAP in Schwann cells of myelinated fibers. Schwann cells from this population also underwent proliferation, indicated by progressive rises in BrdU and GFAP double labeling. Finally, this Schwann cell pool also developed the property of migration, traveling to the distal outgrowth zone, but also with lateral penetration into the perineurium and epineurium, while in intimate contact with new axons. The findings suggest that other signals, in the injured proximal nerve stumps, beyond actual loss of axons, induce 'mature' Schwann cells of myelinated axons to dedifferentiate into those that up-regulated their GFAP expression, proliferate and migrate with axons.
Neuroscience 02/2002; 115(1):321-9. · 3.38 Impact Factor
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ABSTRACT: In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.
Experimental Neurology 02/2002; 173(1):77-85. · 4.70 Impact Factor
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ABSTRACT: The long-term relationship between the peripheral nerve trunk and its vascular supply, the vasa nervorum, has not been considered in the context of denervation and regeneration. While the microvessels of peripheral nerve are not thought to influence Wallerian degeneration itself, in this work we explored how vasa nervorum respond to denervation of the nerve trunk. Our hypotheses were that the presence of axons had a significant impact on the vasa nervorum and that the absence of reinnervation might eventually lead to an unfavorable ischemic regenerative microenvironment. We studied rat sciatic nerve trunks for up to 6 months following transection and either prevented regeneration or allowed it to proceed. Vasa nervorum were studied in several ways: (i) measurements of local endoneurial blood flow using microelectrode hydrogen clearance polarography; (ii) measurements of erythrocyte flux (flow) in the extrinsic nerve plexus using laser Doppler flowmetry; (iii) India ink perfusion of microvessels in unfixed nerve; (iv) mRNA expression of vascular endothelial growth factor (VEGF) using reverse transcription polymerase chain reaction. Early after injury, there were rises in endoneurial and extrinsic flow, microvessel numbers, and VEGF mRNA expression. Angiogenesis was apparently confined to the epineurial and perineurial compartments. Later, however, there were substantial declines in flow observed in long-term (6-month) denervated sciatic nerve trunks associated with declines in the caliber of new microvessels. Reinnervated sciatic nerves had restored endoneurial blood flow. The findings confirm important relationships between axon presence and local blood flow. Angiogenesis is a feature of the injured peripheral nerve, but long term denervated nerve trunks have declines of flow despite retaining new microvessels.
Experimental Neurology 01/2002; 172(2):398-406. · 4.70 Impact Factor
