Visualizing peripheral nerve regeneration by whole mount staining.

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries

Serum Heat Shock Protein 27 and Diabetes Complications in the EURODIAB Prospective Complications Study : A Novel Circulating Marker for Diabetic Neuropathy

OBJECTIVE—Heat shock protein 27 (HSP27) is a member of the small heat shock protein family of proteins. HSP27 expression is enhanced in target tissues of diabetic microvascular complications, and changes in circulating serum HSP27 levels (sHSP27) have been reported in patients with macrovascular disease. We investigated whether sHSP27 levels were associated with micro- and macrovascular complications in type 1 diabetic patients

Caveolin-1 and Altered Neuregulin Signaling Contribute to the Pathophysiological Progression of Diabetic Peripheral Neuropathy

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.OBJECTIVE Evaluate if Erb B2 activation and the loss of caveolin-1 (Cav1) contribute to the pathophysiological progression of diabetic peripheral neuropathy (DPN). RESEARCH DESIGN AND METHODS Cav1 knockout and wild-type C57BL/6 mice were rendered diabetic with streptozotocin, and changes in motor nerve conduction velocity (MNCV), mechanical and thermal hypoalgesia, Erb B2 phosphorylation (pErb B2), and epidermal nerve fiber density were assessed. The contribution of Erb B2 to DPN was assessed using the Erb B2 inhibitors PKI 166 and erlotinib and a conditional bitransgenic mouse that expressed a constitutively active form of Erb B2 in myelinated Schwann cells (SCs). RESULTS Diabetic mice exhibited decreased MNCV and mechanical and thermal sensitivity, but the extent of these deficits was more severe in diabetic Cav1 knockout mice. Diabetes increased pErb B2 levels in both genotypes, but the absence of Cav1 correlated with a greater increase in pErb B2. Erb B2 activation contributed to the mechanical hypoalgesia and MNCV deficits in both diabetic genotypes because treatment with erlotinib or PKI 166 improved these indexes of DPN. Similarly, induction of a constitutively active Erb B2 in myelinated SCs was sufficient to decrease MNCV and induce a mechanical hypoalgesia in the absence of diabetes. CONCLUSIONS Increased Erb B2 activity contributes to specific indexes of DPN, and Cav1 may be an endogenous regulator of Erb B2 signaling. Altered Erb B2 signaling is a novel mechanism that contributes to SC dysfunction in diabetes, and inhibiting Erb B2 may ameliorate deficits of tactile sensitivity in DPN. Diabetic peripheral neuropathy (DPN) is a common complication of diabetes (1). Although hyperglycemia is the definitive cause of DPN (2), the vascular, glial, and neuronal damage that underlies the progressive axonopathy in DPN has a complex biochemical etiology involving oxidative stress (3,4), protein glycation (5), protein kinase C activation (6), polyol synthesis (7), and the hexosamine pathway (8). Altered neurotrophic support also contributes to sensory neuron dysfunction in DPN (9), but whether diabetes may alter growth factor signaling in Schwann cells (SCs), which also undergo substantial degeneration in diabetes, is poorly defined. Neuregulins are growth factors that control SC growth, survival, and differentiation via their interaction with Erb B receptors (10). Although Erb B2 signaling promotes developmental myelination and is clearly trophic for SCs, pharmacological evidence supports that pathologic activation of Erb B2 after axotomy (11) or infection with leprosy bacilli (12) is sufficient to induce SC dedifferentiation and demyelination. Additionally, genetic evidence supports that Erb B2 can promote the development of sensory neuropathies independent of diabetes because expression of a dominant-negative Erb B4 in nonmyelinating (13) or myelinating (14) SCs induced a temperature or mechanical sensory neuropathy, respectively. Given the contribution of Erb B2 to the degeneration of SCs, endogenous proteins that regulate Erb B2 activity may influence the development of certain aspects of sensory neuropathies. The interaction of Erb B2 with the protein caveolin-1 (Cav1) inhibits the intrinsic tyrosine kinase activity of the receptor (15). Cav1 is highly expressed in mature, myelinated SCs (16), and we have shown that prolonged hyperglycemia promoted the downregulation of Cav1 in SCs of sciatic nerve (17). Cav1 may regulate Erb B2 signaling in SCs because its forced downregulation was sufficient to enhance neuregulin-induced demyelination of SC–dorsal root ganglion (DRG) neuron cocultures (18). However, it is unknown whether an increase in Erb B2 activity may contribute to the pathophysiological development of DPN and if changes in Cav1 expression may alter Erb B2 activation in diabetic nerve. In the current study, we demonstrate that diabetic Cav1 knockout mice showed an increased activation of Erb B2 and developed greater motor nerve conduction velocity (MNCV) deficits relative to their wild-type counterparts. Inhibition of Erb B2 with two structurally diverse inhibitors corrected the MNCV deficits and mechanical hypoalgesia evident after 6 or 15 weeks of diabetes. Also, induction of a constitutively active Erb B2 in myelinated SCs of adult mice was sufficient to recapitulate the MNCV and mechanical sensitivity deficits observed in the diabetic mice. These studies provide the first evidence that activation of Erb B2 contributes to deficits associated with myelinated fiber function in diabetic nerve and suggest that Cav1 may serve as an endogenous regulator of Erb B2.This work was supported by grants from the Juvenile Diabetes Research Foundation and the National Institutes of Health (NS-054847 and DK-073594)

Calnexin is necessary for T cell transmigration into the central nervous system

In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS

Drug-mediated inhibition of Fli-1 for the treatment of leukemia

The Ets transcription factor, Fli-1 is activated in murine erythroleukemia and overexpressed in various human malignancies including Ewing's sarcoma, induced by the oncogenic fusion protein EWS/Fli-1. Recent studies by our group and others have demonstrated that Fli-1 plays a key role in tumorigenesis, and disrupting its oncogenic function may serve as a potential treatment option for malignancies associated with its overexpression. Herein, we describe the discovery of 30 anti-Fli-1 compounds, characterized into six functional groups. Treatment of murine and human leukemic cell lines with select compounds inhibits Fli-1 protein or mRNA expression, resulting in proliferation arrest and apoptosis. This anti-cancer effect was mediated, at least in part through direct inhibition of Fli-1 function, as anti-Fli-1 drug treatment inhibited Fli-1 DNA binding to target genes, such as SHIP-1 and gata-1, governing hematopoietic differentiation and proliferation. Furthermore, treatment with select Fli-1 inhibitors revealed a positive relationship between the loss of DNA-binding activity and Fli-1 phosphorylation. Accordingly, anti-Fli-1 drug treatment significantly inhibited leukemogenesis in a murine erythroleukemia model overexpressing Fli-1. This study demonstrates the ability of this drug-screening strategy to isolate effective anti-Fli-1 inhibitors and highlights their potential use for the treatment of malignancies overexpressing this oncogene

Calnexin is necessary for T cell transmigration into the central nervous system.

In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site

Global Transcriptional Programs in Peripheral Nerve Endoneurium and DRG Are Resistant to the Onset of Type 1 Diabetic Neuropathy in Ins2Akita/+ Mice

While the morphological and electrophysiological changes underlying diabetic peripheral neuropathy (DPN) are relatively well described, the involved molecular mechanisms remain poorly understood. In this study, we investigated whether phenotypic changes associated with early DPN are correlated with transcriptional alterations in the neuronal (dorsal root ganglia [DRG]) or the glial (endoneurium) compartments of the peripheral nerve. We used Ins2Akita/+ mice to study transcriptional changes underlying the onset of DPN in type 1 diabetes mellitus (DM). Weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Ins2Akita/+ and control mice during the first three months of life in order to determine the onset of DPN. Based on this phenotypic characterization, we performed gene expression profiling using sciatic nerve endoneurium and DRG isolated from pre-symptomatic and early symptomatic Ins2Akita/+ mice and sex-matched littermate controls. Our phenotypic analysis of Ins2Akita/+ mice revealed that DPN, as measured by reduced MNCV, is detectable in affected animals already one week after the onset of hyperglycemia. Surprisingly, the onset of DPN was not associated with any major persistent changes in gene expression profiles in either sciatic nerve endoneurium or DRG. Our data thus demonstrated that the transcriptional programs in both endoneurial and neuronal compartments of the peripheral nerve are relatively resistant to the onset of hyperglycemia and hypoinsulinemia suggesting that either minor transcriptional alterations or changes on the proteomic level are responsible for the functional deficits associated with the onset of DPN in type 1 DM

Ndel1 Promotes Axon Regeneration via Intermediate Filaments

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration

Analysis of arterial intimal hyperplasia: review and hypothesis

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background: Despite a prodigious investment of funds, we cannot treat or prevent arteriosclerosis and restenosis, particularly its major pathology, arterial intimal hyperplasia. A cornerstone question lies behind all approaches to the disease: what causes the pathology? Hypothesis: I argue that the question itself is misplaced because it implies that intimal hyperplasia is a novel pathological phenomenon caused by new mechanisms. A simple inquiry into arterial morphology shows the opposite is true. The normal multi-layer cellular organization of the tunica intima is identical to that of diseased hyperplasia; it is the standard arterial system design in all placentals at least as large as rabbits, including humans. Formed initially as one-layer endothelium lining, this phenotype can either be maintained or differentiate into a normal multi-layer cellular lining, so striking in its resemblance to diseased hyperplasia that we have to name it "benign intimal hyperplasia". However, normal or "benign " intimal hyperplasia, although microscopically identical to pathology, is a controllable phenotype that rarely compromises blood supply. It is remarkable that each human heart has coronary arteries in which a single-layer endothelium differentiates earl