Search

Studies of neurodegenerative conditions such as Alzheimer’s disease (AD) using post mortem brain tissues have uncovered several perturbations in metals such as copper, iron, and zinc. However, studies of the effects of key, potentially confounding variables on these tissues are currently lacking. Moreover, human-brain tissues have limited availability, further enhancing the difficulty of matching potentially-significant variables including age, sex-matching, post-mortem delay (PMD), and neuropathological stage. This study aimed to investigate the effects of such factors and how they might influence metal concentrations in post-mortem brains. Cingulate gyrus from AD cases and matched controls was obtained from two brain banks, based in Auckland, New Zealand and Manchester, UK. Inductively-coupled plasma mass spectrometry (ICP-MS) was employed to measure levels of nine essential metals in brain tissues, and compared concentrations between cases and controls, and between cohorts, to analyse effects of age, sex, Braak stage, brain weight, and PMD. The same methods were used to investigate the effects of PMD under more controlled conditions using ex vivo healthy adult rat-brain tissue. Metal concentrations in human brain were found to be unmodified by differences in age, sex-matching, Braak stage, brain weight, and PMD between cohorts. Some metals were, however, found to vary significantly across different regions in rat brains. These results indicate that investigations of metal homeostasis in AD and other neurodegenerative conditions can be reliably performed using brain tissues without confounding by varying PMD, age, sex-matching, brain weight, and Braak stage. However, regions of study should be selected carefully.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

Objective The impact of diabetes mellitus on the central nervous system is less widely studied than in the peripheral nervous system, but there is increasing evidence that it elevates the risk of developing cognitive deficits. The aim of this study was to characterize the impact of experimental diabetes on the proteome and metabolome of the hippocampus. We tested the hypothesis that the vitamin B6 isoform pyridoxamine is protective against functional and molecular changes in diabetes. Methods We tested recognition memory using the novel object recognition (NOR) test in streptozotocin (STZ)-induced diabetic, age-matched control, and pyridoxamine- or insulin-treated diabetic male Wistar rats. Comprehensive untargeted metabolomic and proteomic analyses, using gas chromatography-mass spectrometry and iTRAQ-enabled protein quantitation respectively, were utilized to characterize the molecular changes in the hippocampus in diabetes. Results We demonstrated diabetes-specific, long-term (but not short-term) recognition memory impairment and that this deficit was prevented by insulin or pyridoxamine treatment. Metabolomic analysis showed diabetes-associated changes in 13/82 identified metabolites including polyol pathway intermediates glucose (9.2-fold), fructose (4.9-fold) and sorbitol (5.2-fold). We identified and quantified 4807 hippocampal proteins; 806 were significantly altered in diabetes. Pathway analysis revealed significant alterations in cytoskeletal components associated with synaptic plasticity, glutamatergic signaling, oxidative stress, DNA damage and FXR/RXR activation pathways in the diabetic rat hippocampus. Conclusions Our data indicate a protective effect of pyridoxamine against diabetes-induced cognitive deficits, and our comprehensive ‘omics datasets provide insight into the pathogenesis of cognitive dysfunction enabling development of further mechanistic and therapeutic studies., Highlights • Pyridoxamine prevented long-term recognition memory deficits in diabetic rats. • Hippocampal protein changes are revealed by comprehensive untargeted proteomics. • Hippocampal metabolomic analysis showed increased polyol pathway intermediates. • Pyridoxamine altered synaptic plasticity-associated proteins in diabetic rats.

Transforming growth factor‐β (TGFβ) has been reported to be dysregulated in malformed ureters. There exists, however, little information on whether altered TGFβ levels actually perturb ureter development. We therefore hypothesised that TGFβ has functional effects on ureter morphogenesis. Tgfb1, Tgfb2 and Tgfb3 transcripts coding for TGFβ ligands, as well as Tgfbr1 and Tgfbr2 coding for TGFβ receptors, were detected by quantitative polymerase chain reaction in embryonic mouse ureters collected over a wide range of stages. As assessed by in situ hybridisation and immunohistochemistry, the two receptors were detected in embryonic urothelia. Next, TGFβ1 was added to serum‐free cultures of embryonic day 15 mouse ureters. These organs contain immature smooth muscle and urothelial layers and their in vivo potential to grow and acquire peristaltic function can be replicated in serum‐free organ culture. Such organs therefore constitute a suitable developmental stage with which to define roles of factors that affect ureter growth and functional differentiation. Exogenous TGFβ1 inhibited growth of the ureter tube and generated cocoon‐like dysmorphogenesis. RNA sequencing suggested that altered levels of transcripts encoding certain fibroblast growth factors (FGFs) followed exposure to TGFβ. In serum‐free organ culture exogenous FGF10 but not FGF18 abrogated certain dysmorphic effects mediated by exogenous TGFβ1. To assess whether an endogenous TGFβ axis functions in developing ureters, embryonic day 15 explants were exposed to TGFβ receptor chemical blockade; growth of the ureter was enhanced, and aberrant bud‐like structures arose from the urothelial tube. The muscle layer was attenuated around these buds, and peristalsis was compromised. To determine whether TGFβ effects were limited to one stage, explants of mouse embryonic day 13 ureters, more primitive organs, were exposed to exogenous TGFβ1, again generating cocoon‐like structures, and to TGFβ receptor blockade, again generating ectopic buds. As for the mouse studies, immunostaining of normal embryonic human ureters detected TGFβRI and TGFβRII in urothelia. Collectively, these observations reveal unsuspected regulatory roles for endogenous TGFβ in embryonic ureters, fine‐tuning morphogenesis and functional differentiation. Our results also support the hypothesis that the TGFβ up‐regulation reported in ureter malformations impacts on pathobiology. Further experiments are needed to unravel the intracellular signalling mechanisms involved in these dysmorphic responses. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Supplemental Digital Content is Available in the Text. This unique multicenter approach provides high-quality evidence validating burrowing as a robust and reproducible outcome measure to infer the global effect of pain on rodents., Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats (Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP—all animals allocated to treatment; n = 249) and a selected population (SP—TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding “poor” burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of −374 g (−479 to −269 g) for TP and −498 g (−609 to −386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.

Spinal glial cell activation and cytokine secretion have been implicated in the etiology of neuropathic pain in a number of experimental models, including diabetic neuropathy. In this study, streptozotocin- (STZ-) induced diabetic rats were either untreated or treated with gabapentin (50 mg/kg/day by gavage for 2 weeks, from 6 weeks after STZ). At 8 weeks after STZ, hypersensitivity was confirmed in the untreated diabetic rats as a reduced response threshold to touch, whilst mechanical thresholds in gabapentin-treated diabetic rats were no different from controls. Diabetes-associated thermal hypersensitivity was also ameliorated by gabapentin. We performed a cytokine profiling array in lumbar spinal cord samples from control and diabetic rats. This revealed an increase in L-selectin, an adhesion molecule important for neutrophil transmigration, in the spinal cord of diabetic rats but not diabetic rats treated with gabapentin. Furthermore, we found an increase in the number of neutrophils present in the parenchyma of the spinal cord, which was again ameliorated in gabapentin-treated diabetic rats. Therefore, we suggest that dysregulated spinal L-selectin and neutrophil infiltration into the spinal cord could contribute to the pathogenesis of painful diabetic neuropathy.

Introduction The human genome-scale metabolic reconstruction details all known metabolic reactions occurring in humans, and thereby holds substantial promise for studying complex diseases and phenotypes. Capturing the whole human metabolic reconstruction is an on-going task and since the last community effort generated a consensus reconstruction, several updates have been developed. Objectives We report a new consensus version, Recon 2.2, which integrates various alternative versions with significant additional updates. In addition to re-establishing a consensus reconstruction, further key objectives included providing more comprehensive annotation of metabolites and genes, ensuring full mass and charge balance in all reactions, and developing a model that correctly predicts ATP production on a range of carbon sources. Methods Recon 2.2 has been developed through a combination of manual curation and automated error checking. Specific and significant manual updates include a respecification of fatty acid metabolism, oxidative phosphorylation and a coupling of the electron transport chain to ATP synthase activity. All metabolites have definitive chemical formulae and charges specified, and these are used to ensure full mass and charge reaction balancing through an automated linear programming approach. Additionally, improved integration with transcriptomics and proteomics data has been facilitated with the updated curation of relationships between genes, proteins and reactions. Results Recon 2.2 now represents the most predictive model of human metabolism to date as demonstrated here. Extensive manual curation has increased the reconstruction size to 5324 metabolites, 7785 reactions and 1675 associated genes, which now are mapped to a single standard. The focus upon mass and charge balancing of all reactions, along with better representation of energy generation, has produced a flux model that correctly predicts ATP yield on different carbon sources. Conclusion Through these updates we have achieved the most complete and best annotated consensus human metabolic reconstruction available, thereby increasing the ability of this resource to provide novel insights into normal and disease states in human. The model is freely available from the Biomodels database (http://identifiers.org/biomodels.db/MODEL1603150001). Electronic supplementary material The online version of this article (doi:10.1007/s11306-016-1051-4) contains supplementary material, which is available to authorized users.

Diabetic neuropathy is a common secondary complication of diabetes that impacts on patient's health and well-being. Distal axon degeneration is a key feature of diabetic neuropathy, but the pathological changes which underlie axonal die-back are incompletely understood; despite decades of research a treatment has not yet been identified. Basic research must focus on understanding the complex mechanisms underlying changes that occur in the nervous system during diabetes. To this end, tissue culture techniques are invaluable as they enable researchers to examine the intricate mechanistic responses of cells to high glucose or other factors in order to better understand the pathogenesis of nerve dysfunction. This chapter describes the use of in vitro models to study a wide range of specific cellular effects pertaining to diabetic neuropathy including apoptosis, neurite outgrowth, neurodegeneration, activity, and bioenergetics. We consider problems associated with in vitro modeling and future refinement such as use of induced pluripotent stem cells and microfluidic technology.

High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However, our understanding of the molecular mechanisms that cause the marked distal pathology is incomplete. We performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN. We integrated proteomics and metabolomics from the sciatic nerve (SN), the lumbar 4/5 dorsal root ganglia (DRG), and the trigeminal ganglia (TG) of streptozotocin-diabetic and healthy control rats. Even though all tissues showed a dramatic increase in glucose and polyol pathway intermediates in diabetes, a striking upregulation of mitochondrial oxidative phosphorylation and perturbation of lipid metabolism was found in the distal SN that was not present in the corresponding cell bodies of the DRG or the cranial TG. This finding suggests that the most severe molecular consequences of diabetes in the nervous system present in the SN, the region most affected by neuropathy. Such spatial metabolic dysfunction suggests a failure of energy homeostasis and/or oxidative stress, specifically in the distal axon/Schwann cell–rich SN. These data provide a detailed molecular description of the distinct compartmental effects of diabetes on the PNS that could underlie the distal-proximal distribution of pathology.

OBJECTIVE The goal of this study was to characterize glycation adducts formed in both in vivo extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats and in vitro by glycation of laminin and fibronectin with methylglyoxal and glucose. We also investigated the impact of advanced glycation end product (AGE) residue content of ECM on neurite outgrowth from sensory neurons. RESEARCH DESIGN AND METHODS Glycation, oxidation, and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-induced diabetic rat sciatic nerve (3–24 weeks post-STZ) and of laminin and fibronectin that had been glycated using glucose or methylglyoxal were examined by liquid chromatography with tandem mass spectrometry. Methylglyoxal-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration. RESULTS STZ-induced diabetes produced a significant increase in early glycation Nε-fructosyl-lysine and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by methylglyoxal and glucose increased glycation adduct residue contents with methylglyoxal-derived hydroimidazolone and Nε-fructosyl-lysine, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite outgrowth, which was prevented by aminoguanidine and nerve growth factor. CONCLUSIONS Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.

Neurons have a constantly high glucose demand, and unlike muscle cells they cannot accommodate episodic glucose uptake under the influence of insulin. Neuronal glucose uptake depends on the extracellular concentration of glucose, and cellular damage can ensue after persistent episodes of hyperglycaemia--a phenomenon referred to as glucose neurotoxicity. This article reviews the pathophysiological manifestation of raised glucose in neurons and how this can explain the major components of diabetic neuropathy.

The effect of streptozotocin (STZ)-induced diabetes on expression and activity of hexokinase, the first enzyme and rate-limiting step in glycolysis, was studied in sensory neurons of lumbar dorsal root ganglia (DRG). The DRG and sciatic nerve of adult rats expressed the hexokinase I isoform only. Immunofluorescent staining of lumbar DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large, mainly proprioceptive neurons, had very low or negative staining for hexokinase I. Intracellular localization and biochemical studies on intact DRG from adult rats and cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. Duration of STZ-diabetes of 6 or 12 weeks diminished hexokinase activity by 28% and 30%, respectively, in lumbar DRG compared with age matched controls (P

Diabetic neuropathy is a major complication of diabetes and has multifactoral aetiology. The exact cause of damage is unknown although high glucose and oxidative stress are known to contribute significantly. In order to identify molecular targets of the disease and possibly new therapeutic targets, we previously examined the effect of diabetes on dorsal root ganglia (DRG) neurons using Affymetrix gene chip arrays. A number of individual genes and groups of genes were found to be dysregulated; the most significant of these was thioredoxin interacting protein (Txnip). This gene was found to have increased expression in DRG from diabetic rats with all durations of diabetes examined, including those that preceded the onset of functional changes such as decreased nerve conduction velocity. Increased Txnip expression therefore represents an early change in diabetic neuropathy that could, at least in part, be responsible for causing the initial functional deficits. This study confirmed the changes in Txnip expression at the mRNA and protein levels and identified the cell types responsible for the change. Furthermore we investigated the mechanism of diabetes-induced Txnip gene induction. Neither the antioxidant dexlipotam (R-lipoic acid) nor the p38 MAP kinase inhibitor SB239063 could prevent increases in Txnip expression despite reducing oxidative stress. However, treatment of rats with insulin prevented diabetes-induced increases in Txnip gene expression. These results indicate another mechanism by which diabetes may cause oxidative damage in peripheral nerve, and may represent a novel target for therapeutic intervention.

The successful regeneration of peripheral branches of sensory neurons following injury is attributed to the presence of neurotrophins and interaction of regenerating axons with the extracellular matrix. Here, we show that the laminin receptor, alpha7beta1 integrin is a crucial mediator of neurite outgrowth from distinct populations of sensory neurons. Following sciatic nerve crush, alpha7 integrin is expressed by medium-large diameter, NF200-immunoreactive (IR), and medium diameter, CGRP-IR, neurons, but very few small diameter non-peptidergic neurons. The functional significance of alpha7 integrin expression following injury was addressed using dissociated adult rat and mouse sensory neurons. By using function-blocking antibodies and neurons isolated from alpha7 integrin null mice, we demonstrate that NGF- and NT-3-stimulated neurite outgrowth is reduced in the absence of alpha7 integrin signaling. In contrast, GDNF-stimulated neurite outgrowth is less dependent on alpha7 integrin. These results define an essential interaction between alpha7 integrin and laminin for mediating neurite outgrowth of subpopulations of injured adult sensory neurons.

Regeneration of injured adult sensory neurons within the CNS is essentially abortive, attributable in part to lesion-induced or revealed inhibitors such as the chondroitin sulfate proteoglycans and the myelin inhibitors (Nogo-A, MAG, and OMgp). Much of this inhibition may be overcome by boosting the growth status of sensory neurons by delivering a conditioning lesion to their peripheral branches. Here, we identify a key role for the lesion-induced cytokine interleukin-6 (IL-6) in mediating conditioning lesion-induced enhanced regeneration of injured dorsal column afferents. In adult mice,conditioning injury to the sciatic nerve 1 week before bilateral dorsal column crush resulted in regeneration of dorsal column axons up to and beyond the injury site into host CNS tissue. This enhanced growth state was accompanied by an increase in the expression of the growth-associated protein GAP43 in preinjured but not intact dorsal root ganglia (DRGs). Preconditioning injury of the sciatic nerve in IL-6 –/– mice resulted in the total failure in regeneration of dorsal column axons consequent on the lack of GAP43 upregulation after a preconditioning injury. DRGs cell counts and cholera toxin β subunit labeling revealed that impaired regeneration in knock-out mice was unrelated to cell loss or a deficit in tracer transport.In vitro, exogenous IL-6 boosted sensory neuron growth status as evidenced by enhanced neurite extension. This effect required NGF or NT-3 but not soluble IL-6 receptor as cofactors. Evidence of conditioning lesion-enhanced growth status of DRGs cells can also be observedin vitroas an earlier and enhanced rate of neurite extension; this phenomenon fails in IL-6 –/– mice preinjured 7 din vivo. We suggest that injury-induced IL-6 upregulation is required to promote regeneration within the CNS. Our results indicate that this is achieved through a boosted growth state of dorsal column projecting sensory neurons.

Members of the interleukin-6 (IL-6) family of cytokines have been implicated as major mediators of the response of the adult nervous system to injury. The basis for an interaction of IL-6 cytokines with adult sensory neurones has been established by analysing the levels and distribution of the two signal-transducing receptor subunits, glycoprotein 130 (gp130) and leukaemia inhibitory factor receptor (LIFR), in the dorsal root ganglion (DRG) of male adult rats before and following nerve injury. All sensory neurones express gp130-immunoreactivity (IR) in the cytoplasm and on the plasma membrane. Levels of gp130 and its intracellular distribution remained unchanged up to 14 days following sciatic nerve axotomy. LIFR-IR was largely absent from the cytoplasm and plasma membrane of sensory neurones, but confined almost exclusively to the nuclear compartment. However, following axotomy, punctate cytoplasmic LIFR-IR was detected which persisted up to 28 days following axotomy. The expression of cytoplasmic LIFR 2 days post-axotomy was proportionally greater in a subset of small diameter sensory neurones which expressed either the sensory neuropeptide CGRP or the cell surface marker isolectin B4. The coexpression of gp130 and LIFR in the same intracellular compartment following axotomy conveys potential responsiveness of injured sensory neurones to members of the IL-6 family of cytokines.

Minocycline is an inhibitor of matrix metalloproteinases (MMPs) and has been shown to have analgesic effects. Whilst increased expression of MMPs is associated with neuropathic pain, MMPs also play crucial roles in Wallerian degeneration and nerve regeneration. In this study we examined the expression of MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1/-2 in the sciatic nerve of control and streptozotocin-induced diabetic rats treated with either vehicle or minocycline by quantitative PCR and gelatin zymography. We assessed the effects of minocycline on nerve conduction velocity and intraepidermal nerve fibre (IENF) deficits in diabetic neuropathy and investigated the effects of minocycline or MMP-2 on neurite outgrowth from primary cultures of dissociated adult rat sensory neurons. We show that MMP-2 is expressed constitutively in the sciatic nerve in vivo and treatment with minocycline or diabetes leads to downregulation of MMP-2 expression and activity. The functional consequence of this is IENF deficits in minocycline-treated nondiabetic rats and an unsupportive microenvironment for regeneration in diabetes. Minocycline reduces levels of MMP-2 mRNA and nerve growth factor-induced neurite outgrowth. Furthermore, in vivo minocycline treatment reduces preconditioning-induced in vitro neurite outgrowth following a sciatic nerve crush. In contrast, the addition of active MMP-2 facilitates neurite outgrowth in the absence of neurotrophic support and pre-treatment of diabetic sciatic nerve substrata with active MMP-2 promotes a permissive environment for neurite outgrowth. In conclusion we suggest that MMP-2 downregulation may contribute to the regenerative deficits in diabetes. Minocycline treatment also downregulates MMP-2 activity and is associated with inhibitory effects on sensory neurons. Thus, caution should be exhibited with its use as the balance between beneficial and detrimental outcomes may be critical in assessing the benefits of using minocycline to treat diabetic neuropathy., Highlights • MMP-2, but not MMP-9, is constitutively expressed in the adult rat sciatic nerve. • Levels of cleaved active MMP-2 are reduced in sciatic nerve of diabetic rats. • Active MMP-2 potentiates neurite outgrowth from sensory neurons. • Minocycline reduces levels of MMP-2 mRNA and impairs NGF-induced neurite growth. • Minocycline did not prevent nerve dysfunction in experimental diabetic neuropathy.

Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.

The somatosensory nervous system is responsible for the transmission of a multitude of sensory information from specialized receptors in the periphery to the central nervous system. Sensory afferents can potentially be damaged at several sites: in the peripheral nerve; the dorsal root; or the dorsal columns of the spinal cord; and the success of regeneration depends on the site of injury. The regeneration of peripheral nerve branches following injury is relatively successful compared to central branches. This is largely attributed to the presence of neurotrophic factors and a Schwann cell basement membrane rich in permissive extracellular matrix (ECM) components which promote axonal regeneration in the peripheral nerve. Modulation of the ECM environment and/or neuronal integrins may enhance regenerative potential of sensory neurons following peripheral or central nerve injury or disease. This review describes the interactions between integrins and ECM molecules (particularly the growth supportive ligands, laminin, and fibronectin; and the growth inhibitory chondroitin sulfate proteoglycans (CSPGs)) during development and regeneration of sensory neurons following physical injury or neuropathy.

In Type 1 and 2 diabetes tissue stiffening is evident from measurements of the gross mechanical properties of the vasculature. Elastic fibers play an important role in the mechanical function of vascular tissue, however, the effects of diabetes on individual elastic fiber components remains poorly defined. Fibrillin microfibrils, a key elastic fiber component, have a ‘beads-on-a-string’ structure with a periodicity of approximately 56 nm. We tested for possible disruption due to diabetes in fibrillin microfibrils isolated from rat aorta using an experimental model of Type 1 diabetes in rats. The isolated fibrillin microfibrils were imaged with atomic force microscopy (AFM) and image analysis techniques were used to characterise the microfibrils. Although there was no significant difference in mean microfibril length (control 23.2 repeats, SEM 6.2 repeats: diabetic 23.6 repeats, SEM 6.1 repeats: Mann Whitney U-test, p=0.391), mean periodicity was significantly reduced in microfibrils isolated from the diabetic rats (52.7 nm, SEM 0.4 nm) compared with age-matched controls (59.5 nm, SEM 0.4 nm) (p

OBJECTIVE The objectives of the study were to evaluate retrograde axonal transport of vascular endothelial growth factor A (VEGF-A) protein to sensory neurons after intramuscular administration of an engineered zinc finger protein activator of endogenous VEGF-A (VZ+434) in an experimental model of diabetes, and to characterize the VEGF-A target neurons. RESEARCH DESIGN AND METHODS We compared the expression of VEGF-A in lumbar (L)4/5 dorsal root ganglia (DRG) of control rats and VZ+434-treated and untreated streptozotocin (STZ)-induced diabetic rats. In addition, axonal transport of VEGF-A, activation of signal transduction pathways in the DRG, and mechanical sensitivity were assessed. RESULTS VEGF-A immunoreactivity (IR) was detected in small- to medium-diameter neurons in DRG of control rats. Fewer VEGF-A-IR neurons were observed in DRG from STZ-induced diabetic rats; this decrease was confirmed and quantified by Western blotting. VZ+434 administration resulted in a significant increase in VEGF-A protein expression in ipsilateral DRG, 24 h after injection. VEGF-A was axonally transported to the DRG via the sciatic nerve. VZ+434 administration resulted in significant activation of AKT in the ipsilateral DRG by 48 h that was sustained for 1 week after injection. VZ+434 protected against mechanical allodynia 8 weeks after STZ injection. CONCLUSIONS Intramuscular administration of VZ+434 increases VEGF-A protein levels in L4/5 DRG, correcting the deficit observed after induction of diabetes, and protects against mechanical allodynia. Elevated VEGF-A levels result from retrograde axonal transport and are associated with altered signal transduction, via the phosphatidylinositol 3′-kinase pathway. These data support a neuroprotective role for VEGF-A in the therapeutic actions of VZ+434 and suggest a mechanism by which VEGF-A exerts this activity.

This review examines the putative role of glucose in the etiology of diabetic neuropathies. Excessive glucose generates several secondary metabolic anomalies - principally oxidative stress (via both the polyol pathway and glucoxidation) and non-enzymic glycation of macromolecules. The latter is also facilitated by glucoxidation. These metabolic deviations trigger cellular responses that are inappropriate to normal function. Principal among these are neurotrophic deficits and phosphorylation of mitogen-activated protein kinases (MAPK). Downstream of these events are aberrant ion channel function and disordered gene expression, leading to changes in cellular phenotype. This leads directly to disordered nerve conduction, a recognised early clinical sign, and indirectly, via as yet undisclosed links, to sensory loss and axonopathy. Recent work also links MAPK activation to the development of neuropathic pain.

Hexokinase is known as the first enzyme and rate-limiting step in glycolysis. The role of hexokinase activity and localization in regulating the rate of axonal regeneration was studied in cultured adult sensory neurons of dorsal root ganglia (DRG). Immunofluorescent staining of DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large neurons had negative staining for hexokinase I. Intracellular localization and biochemical studies in cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. The hypothesis that neurotrophic factor dependent activation of Akt would regulate hexokinase association with the mitochondria was tested and quantitative Western blotting showed no effect of blockade of the phosphoinositide 3-kinase (PI 3-kinase)/Akt pathway using the inhibitor LY294002, indicating this interaction of hexokinase with mitochondria was not neurotrophic factor or Akt-dependent. Finally, pharmacological blockade of hexokinase activity and inhibition of localization to the mitochondrial compartment with hexokinase II VDAC binding domain (Hxk2VBD) peptide caused a significant inhibition of neurotrophic factor-directed axon outgrowth. The results support a key role for hexokinase activity and/or localization to the mitochondria in the regulation of neurite outgrowth in cultured adult sensory neurons.

OBJECTIVE—Axonal regeneration is defective in both experimental and clinical diabetic neuropathy, contributing to loss of axonal extremities and neuronal dysfunction. The mechanisms behind this failure are not fully understood; however, a deficit in neurotrophic support and signaling has been implicated. RESEARCH DESIGN AND METHODS—We investigated the expression of neuritin (also known as candidate plasticity gene 15, cpg15) in the sensory nervous system of control rats and rats with streptozotocin (STZ)-induced diabetes using microarray PCR, Western blotting, and immunocytochemical analysis. The functional role of neuritin in sensory neurons in vitro was assessed using silencing RNA. RESULTS—Neuritin was expressed by a population of small-diameter neurons in the dorsal root ganglia (DRG) and was anterogradely and retrogradely transported along the sciatic nerve in vivo. Nerve growth factor (NGF) treatment induced an increase in the transcription and translation of neuritin in sensory neurons in vitro. This increase was both time and dose dependent and occurred via mitogen-activated protein kinase or phosphatidylinositol-3 kinase activation. Inhibition of neuritin using silencing RNA abolished NGF-mediated neurite outgrowth, demonstrating the crucial role played by neuritin in mediating regeneration. Neuritin levels were reduced in both the DRG and sciatic nerve of rats with 12 weeks of STZ-induced diabetes, and these deficits were reversed in vivo by treatment with NGF. CONCLUSIONS—Manipulation of neuritin levels in diabetes may therefore provide a potential target for therapeutic intervention in the management of neuropathy.

We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.

Embryonic dorsal root ganglion (DRG) neurons die after axonal damage in vivo, and cultured embryonic DRG neurons require exogenous neurotrophic factors that activate the neuroprotective transcription factor nuclear factor-kappaB(NF-kappaB) for survival. In contrast, adult DRG neurons survive permanent axotomy in vivo and in defined culture media devoid of exogenous neurotrophic factors in vitro. Peripheral axotomy in adult rats induces local accumulation of the cytokine tumor necrosis factor alpha( TNFalpha), a potent activator of NF-kappaB activity. We tested the hypothesis that activation of NF-kappaB stimulated by endogenous TNFalpha was required for survival of axotomized adult sensory neurons. Peripheral axotomy of lumbar DRG neurons by sciatic nerve crush induced a very rapid ( within 2 h) and significant elevation in NF-kappaB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-kappaB nuclear translocation and a significant rise in NF-kappaB DNA-binding activity after plating. Inhibitors of NF-kappaB ( SN50 or NF-kappaB decoy DNA) resulted in necrotic cell death of medium to large neurons (greater than or equal to 40 mum) within 24 h ( 60 and 75%, respectively), whereas inhibition of p38 and mitogen-activated protein/extracellular signal-regulated kinase did not effect survival. ELISA revealed that these cultures contained TNFalpha, and exposure to an anti-TNFalpha antibody inhibited NF-kappaB DNA-binding activity by similar to35% and killed similar to40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-kappaB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.

This study tested the premise that immunoreactivity representing the p75 neurotrophin receptor (p75(NTR)) appears in plasma of diabetic rats in association with the early stages of neuronal dysfunction or damage. We also examined whether treatment beneficial to neuropathy might reduce the p75(NTR) immunoreactivity.Plasma proteins were fractionated by SDS-PAGE and immunoblots exposed to p75(NTR) antibody, using receptor protein from cultured PC12 cells as an external standard. Rats were made diabetic with streptozotocin for various periods and exsanguinated. Plasma glucose, HbA(1)c and plasma proteins were determined. We also studied plasma samples from diabetic mice lacking the gene coding for p75(NTR), as well as the effect of sciatic nerve crush on healthy male Wistar rats.Plasma p75(NTR) immunoreactivity began to exceed normal levels at 8 weeks after induction of diabetes, and was significantly raised at 10 (p0.05) and 12 weeks (p0.001). Treatment between 8 and 12 weeks with insulin, fidarestat (an aldose reductase inhibitor), nerve growth factor and neurotrophin 3 all normalised the plasma p75(NTR) immunoreactivity. Plasma from p75(NTR) (-/-) mice contained no such immunoreactivity, though it was present in plasma from wild-type mice. Following nerve crush, p75(NTR) immunoreactivity appeared in plasma of non-diabetic mice, indicating that this can be a result of nerve trauma.These observations suggest that plasma p75(NTR) immunoreactivity may serve as an early indicator of neuronal dysfunction or damage in diabetes. The time course of its appearance relates well to that of early neuropathy and its response to interventions that are neuroprotective suggests that it might mirror neurological status.

Conditioning injury to adult mammalian sensory neurons enhances their regeneration potential. Here we show that leukemia inhibitory factor (LIF) is a fundamental component of the conditioning response. Conditioning injury in vivo significantly increases the intrinsic growth capacity of sensory neurons in vitro from LIF+/+ mice. This conditioning effect is significantly blunted in sensory neurons from LIF−/− mice. Enhanced growth is rescued in vitro in LIF−/− mice by the addition of exogenous LIF, and the effect blocked by human LIF-05, an LIF receptor antagonist. Furthermore, we demonstrate that LIF promotes elongating but not arborizing neurite outgrowth in vitro and is required for normal regeneration of injured adult sensory neurons in vivo . LIF is also functionally protective to peptidergic sensory neurons after nerve damage in vivo . Our results indicate that the alteration in intrinsic growth status of injured sensory neurons depends, at least in part, on LIF.

In anaesthetized rats, the intraspinal release of immunoreactive prostaglandin E2 was measured using antibody microprobes. We addressed the question of whether the release of immunoreactive prostaglandin E2 is altered during development of acute inflammation in the knee evoked by intra-articular injections of kaolin and carrageenan. We also examined cyclo-oxygenase-1 and cyclo-oxygenase-2 protein levels in the spinal cord during the development of inflammation using the same model of arthritis. Densitometric analysis of microprobes showed that basal release of immunoreactive prostaglandin E2 in the period 175–310 min after kaolin was slightly higher than in the absence of inflammation. A pronounced enhancement of basal release of immunoreactive prostaglandin E2 was observed 430–530 min after kaolin. Enhanced levels of immunoreactive prostaglandin E2 were observed throughout the dorsal and ventral horns. Release of immunoreactive prostaglandin E2 was not altered further by the application of innocuous and noxious pressure onto the inflamed knee. Western blot analysis revealed that cyclo-oxygenase-2 but not cyclo-oxygenase-1 protein levels were elevated in the spinal cords of animals with inflammation compared to normal animals. This effect was evident as early as 3 h after the induction of arthritis. The maximum elevation of cyclo-oxygenase-2 protein levels (six-fold) was observed 12 h after the induction of arthritis. The results show that there is a tonic release of immunoreactive prostaglandin E2 from the spinal cord following the induction of arthritis, which is accompanied by enhanced expression of cyclo-oxygenase-2 protein in the spinal cord. We suggest that intraspinal prostaglandins may play a role in inflammation-evoked central sensitizatlon of spinal cord neurons.

In recent years several studies have examined whether eicosanoids might facilitate neurotransmission in pain pathways. Discrepancies between the doses of some non-steroidal antiinflammatory drugs (NSAIDs) required to produce antiinflammatory and analgesic effects suggest that the analgesic actions may arise through a central action of NSAIDs (McCormack and Urquart, 1995). Evidence supporting this hypothesis has come from behavioural experiments which have shown that intrathecally applied NSAIDs reduce pain-related behaviour in the rat formalin model of inflammation (Malmberg and Yaksh, 1992). Furthermore, NSAIDs applied intravenously reduce the responses of rat spinal cord neurones, rendered hyperexcitable by an acute arthritic lesion, to innocuous and noxious stimuli applied to the inflamed knee (Neugebauer et al., 1995).

In Type 1 and 2 diabetes tissue stiffening is evident from measurements of the gross mechanical properties of the vasculature. In general, pathological glycosylation of extracellular matrix proteins may play an important role in increasing stiffness in diabetic patients. However, the effects of diabetes on individual elastic fibre components remain poorly defined. Fibrillin microfibrils, a key elastic fibre component, have a ‘beads-on-a-string’ structure with a periodicity of approximately 56nm. We tested for possible disruption due to diabetes in fibrillin microfibrils isolated from rat aorta. Diabetes was induced in 250g adult Wistar rats by streptozotocin (STZ) injection (55mg/kg) and were sacrificed 8 weeks later along with age-matched controls. At sacrifice, the STZ-treated rats had severe hyperglycaemia (+/− 28mmol/l). Fibrillin microfibrils were isolated and purified by well-established bacterial collagenase digestion and size-exclusion chromatography prior to imaging with atomic force microscopy. Initial experiments show that fibrillin microfibril periodicity is reduced following STZ treatment; 52.0 +/− 0.4nm (STZ) vs 56.9 +/− 0.4nm (Control), n=600 periodicity measurements, 2 animals per group), (p