Intraoperative visualization of nerves using a myelin protein-zero specific fluorescent tracer.

BACKGROUND: Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future surgery, a fluorescent tracer was developed that specifically targets myelin protein zero (P0). RESULTS: Truncated homotypic P0 protein-based peptide sequences were C-terminally functionalized with the far-red cyanine dye Cy5. The lead compound Cy5-P0101-125 was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0101-125 (KD = 105 ± 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0101-125  enabled in vivo fluorescence imaging of the Sciatic nerve in mice after local intravenous (i.v.) administration and showed compatibility with a clinical fluorescence laparoscope during evaluation in a porcine model undergoing robot-assisted surgery. Biodistribution data revealed that i.v. administered [111In]In-DTPA-P0101-125 does not enter the central nervous system (CNS). CONCLUSION: P0101-125 has proven to be a potent nerve-specific agent that is able to target P0/myelin under in vitro, ex vivo, and in vivo conditions without posing a threat for CNS-related toxicity.

Imaging axon regeneration within synthetic nerve conduits.

While axons within the central nervous system (CNS) do not regenerate following injury, those in the peripheral nervous system (PNS) do, although not in a clinically satisfactory manner as only a small proportion of axons exhibit long-distance regeneration. Moreover, functional recovery is hampered by excessive axonal sprouting and aberrant reinnervation of target tissue. In order to investigate the mechanisms governing the regrowth of axons following injury, previous studies have used lesion paradigms of peripheral nerves in rat or mouse models, and reagents or cells have been administered to the lesion site through nerve conduits, aiming to improve early-stage regeneration. Morphological analysis of such in vivo experiments has however been limited by the incompatibility of synthetic nerve conduits with existing tissue-clearing and imaging techniques. We present herein a novel experimental approach that allows high-resolution imaging of individual axons within nerve conduits, together with quantitative assessment of fiber growth. We used a GFP-expressing mouse strain in a lesion model of the sciatic nerve to describe a strategy that combines nerve clearing, chemical treatment of chitosan nerve conduits, and long working distance confocal microscopy with image processing and analysis. This novel experimental setup provides a means of documenting axon growth within the actual conduit during the critical initial stage of regeneration. This will greatly facilitate the development and evaluation of treatment regimens to improve axonal regeneration following nerve damage.

Nociceptive DRG neurons express muscle lim protein upon axonal injury.

Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

Growth/differentiation factor-15 and its role in peripheral nervous system lesion and regeneration.

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor beta (TGF-ß) superfamily. It is widely distributed in the nervous system, where it has been shown to play an important role in neuronal maintenance. The present study investigates the role of endogenous GDF-15 in sciatic nerve (SN) lesions using wild-type (WT) and GDF-15 knock-out (KO) mice. SN of 5-6-month-old mice were crushed or transected. Dorsal root ganglia (DRG) and nerve tissue were analyzed at different time points from 6 h to 9 weeks post-lesion. Both crush and transection induced GDF-15 mRNA and protein in the distal portion of the nerve, with a peak at day 7. DRG neuron death did not significantly differ between the genotypes; similarly, remyelination of regenerating axons was not affected by the genotype. Alternative macrophage activation and macrophage recruitment were more pronounced in the KO nerve. Protrusion speed of axons was similar in the two genotypes but WT axons showed better maturation, as indicated by larger caliber at 9 weeks. Furthermore, the regenerated WT nerve showed better performance in the electromyography test, indicating better functional recovery. We conclude that endogenous GDF-15 is beneficial for axon regeneration following SN crush.

Extrinsic cellular and molecular mediators of peripheral axonal regeneration.

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.

SDF-1/CXCL12: its role in spinal cord injury.

The chemokine stromal cell-derived factor 1 (SDF-1/CXCL12), is not only the most ancient, but also one of the most potent chemotactic factors. Orchestrating the migration of cells as well as promoting axon outgrowth in the presence of myelin inhibitors, SDF-1 is fundamental to central nervous system development, homeostasis and traumatic injury. SDF-1 attracts endogenous stem/precursor cells and immune cells to the injury site and, upon local infusion, enhances axonal sprouting following spinal cord injury. Together these features make SDF-1 a very exciting molecule for spinal cord repair.

Cortical gene expression in spinal cord injury and repair: insight into the functional complexity of the neural regeneration program.

Traumatic spinal cord injury (SCI) results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005) that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons, and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST). In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1-60 days post-operation (dpo). We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection, and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons toward enhanced cell maintenance and axonal regeneration.

The HOX Code as a "biological fingerprint" to distinguish functionally distinct stem cell populations derived from cord blood.

Mesenchymal stem cells (MSC) have been isolated from almost every adult tissue. In cord blood (CB), different non-hematopoietic CD45-, CD34- adherent cell populations can be generated: the cord blood derived MSC (CB-MSC), that behave almost like MSC from bone marrow (BM-MSC), and unrestricted somatic stem cells (USSC) which show a distinct differentiation potential into all three germ layers. However, distinguishing these populations easily by molecular markers is still a concern. In this study we were able to present the HOX expression pattern of USSC, CB-MSC and BM-MSC, which in fact allows a discrimination of these populations. Briefly, RT-PCR analysis of the HOX code revealed a high similarity between BM-MSC and CB-MSC, which are both HOX-positive, whereas USSC resembled H9 embryonic stem cells HOX-negative.Especially HOXA9, HOXB7, HOXC10 and HOXD8 are good candidate markers to discriminate MSC from USSC. Thus, our data suggest that the "biological fingerprint" based on the HOX code can be used to distinguish functionally distinct MSC populations derived from bone marrow and cord blood.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

SDF-1 stimulates neurite growth on inhibitory CNS myelin.

Impaired axonal regeneration is a common observation after central nervous system (CNS) injury. The stromal cell-derived factor-1, SDF-1/CXCL12, has previously been shown to promote axonal growth in the presence of potent chemorepellent molecules known to be important in nervous system development. Here, we report that treatment with SDF-1alpha is sufficient to overcome neurite outgrowth inhibition mediated by CNS myelin towards cultured postnatal dorsal root ganglion neurons. While we found both cognate SDF-1 receptors, CXCR4 and CXCR7/RDC1, to be coexpressed on myelin-sensitive dorsal root ganglion neurons, the distinct expression pattern of CXCR4 on growth cones and branching points of neurites suggests a function of this receptor in chemokine-mediated growth promotion and/or arborization. These in vitro findings were further corroborated as local intrathecal infusion of SDF-1 into spinal cord injury following thoracic dorsal hemisection resulted in enhanced sprouting of corticospinal tract axons into white and grey matter. Our findings indicate that SDF-1 receptor activation might constitute a novel therapeutic approach to promote axonal growth in the injured CNS.

Gene expression profiling reveals that peripheral nerve regeneration is a consequence of both novel injury-dependent and reactivated developmental processes.

One of the most striking features of the injured mature peripheral nervous system is the ability to regenerate. The lesioned peripheral nervous system displays stereotypic histopathological reactions indicating the activation of a co-ordinated lesion-induced gene expression programme. Previous research has already identified molecular components of this axonal switch from a mature transmitting to a regenerative growth mode. The observed alterations in gene expression within the lesioned distal nerve stump were largely attributed to recapitulated developmental processes. However, to our knowledge, this hypothesis has not been proven systematically. Most of the stereotypic molecular and cellular reactions during nerve development and repair can be assigned to specific time windows. Consequently, we have compared gene expression profiles of both paradigms at six different time-points each by means of cDNA array hybridization. Our data identified injury-specific molecular reactions and revealed to what extent developmental mechanisms are reactivated in response to nerve lesion. Ninety-one genes (47% of the regeneration-associated genes) were found to be significantly regulated in both paradigms, suggesting that regeneration only partially recapitulates development and that approximately half of the regulated genes are part of a regeneration-dependent programme. Interestingly, mainly genes encoding signal transducers or factors involved in processes such as cell death, immune response, transport and transcriptional regulation showed injury-specific gene expression.

Peripheral myelin protein 22 kDa and protein zero: domain specific trans-interactions.

The peripheral myelin proteins P0 and PMP22 are associated in preparations of compact myelin and in cell cultures coexpressing both molecules. The mechanism of this interaction, however, still needs to be unravelled. We have established three different (cell-cell, cell-protein, protein-protein based) assay systems using retrovirally transduced HeLa cells that overexpressed either PMP22 or P0 and purified GST fusion oligopeptides of PMP22 and P0 to detect domain-specific interactions between these proteins. The results revealed that PMP22 and P0 are involved in both trans-homophilic and trans-heterophilic interactions. Moreover, the data clearly indicate that the heterophilic trans-interaction is mediated through the second loop of PMP22, while the first loop is involved in homophilic trans-interaction of PMP22 proteins. Both modes of interaction are due to direct protein-protein binding. In addition, we demonstrate that disease-related point mutations of P0 resulted in a decreased adhesion capability correlating with the severity of the respective disease phenotype.

Identification and characterization of ZFP-57, a novel zinc finger transcription factor in the mammalian peripheral nervous system.

To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells. Its mRNA expression profile within embryonic and adult normal and transected peripheral nerves, and its distribution in the rest of the nervous system is described. High levels of expression are seen in embryonic nerves and spinal cord. These drop rapidly during the first few weeks after birth, a pattern mirrored in other parts of the nervous system. ZFP-57 localizes to the nucleus of Schwann and other cells. The sequence contains an N-terminal Krüppel-associated box (KRAB) domain and ZFP-57 constructs containing green fluorescent protein reveal that the protein colocalizes with heterochromatin protein 1alpha to centromeric heterochromatin in a characteristic speckled pattern in NIH3T3 cells. The KRAB domain is required for this localization, because constructs lacking it target the protein to the nucleus but not to the centromeric heterochromatin. When fused to a heterologous DNA binding domain, the KRAB domain of ZFP-57 represses transcription, and full-length ZFP-57 represses Schwann cell transcription from myelin basic protein and P(0) promoters in co-transfection assays. Zfp-57 mRNA is up-regulated in Schwann cells in response to leukemia inhibitory factor and fibroblast growth factor 2.

Proteolipid plasmolipin: localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin.

The proteolipid plasmolipin is member of the expanding group of tetraspan (4TM) myelin proteins. Initially, plasmolipin was isolated from kidney plasma membranes, but subsequent northern blot analysis revealed highest expression in the nervous system. To gain more insight into the functional roles of plasmolipin, we have generated a plasmolipin-specific polyclonal antibody. Immunohistochemical staining confirms our previous observation of glial plasmolipin expression and proves plasmolipin localization in the compact myelin of rat peripheral nerve and myelinated tracts of the CNS. Western blot analysis indicates a strong temporal correlation of plasmolipin expression and (re-) myelination in the PNS and CNS. However, following axotomy plasmolipin expression is also recovered in non-regenerating distal nerve stumps. In addition, we detected plasmolipin expression in distinct neuronal subpopulations of the CNS. The observed asymmetric distribution of plasmolipin in compact myelin, as well as in epithelial cells of kidney and stomach, indicates a polarized cellular localization. Therefore, we purified myelin from the CNS and PNS and demonstrated an enrichement of phosphorylated plasmolipin protein in detergent-insoluble lipid raft fractions, suggesting selective targeting of plasmolipin to the myelin membranes. The present data indicate that the proteolipid plasmolipin is a structural component of apical membranes of polarized cells and provides the basis for further functional analysis.

Osteopontin: a novel axon-regulated Schwann cell gene.

Osteopontin (OPN) is a RGD-containing glycoprotein with cytokine-like, chemotactic, and pro-adhesive properties. During wound healing, OPN is abundantly expressed by infiltrating macrophages and has been implicated in posttraumatic tissue repair. To delineate a role in the regenerative response to axotomy we examined the expression of OPN in Wallerian degeneration of the sciatic nerve in rats. Unexpectedly, we found high constitutive expression of OPN by myelinating Schwann cells (SCs) in uninjured control nerves. OPN mRNA expression was confirmed in primary cultures of rat SCs. Upon axotomy, SC-expressed OPN in the degenerating distal nerve stump transiently increased during the first days after injury, but was continuously downregulated thereafter, reaching its minimum at Day 14. Macrophages invading axotomized nerves were OPN-negative. During late stages after axotomy, SC-OPN was reexpressed in regenerating but not permanently transected nerves. We also found OPN expression by myelinating SCs in human sural nerves with a dramatic reduction in severe axonal polyneuropathies. Taken together, our study identifies OPN as a novel Schwann cell gene regulated by axon-derived signals. The lack of OPN induction in infiltrating macrophages indicates fundamental differences in tissue repair between axonal injury in the peripheral nervous system and structural lesions in other organ systems.