Local FK506 dose-dependent study using a novel three-dimensional organotypic assay

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG–ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.     

Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.     

LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

CaMKK-CaMK1a,a New Post-Traumatic Signalling Pathway Induced in Mouse Somatosensory Neurons

Neurons innervating peripheral tissues display complex responses to peripheral nerve injury. These include the activation and suppression of a variety of signalling pathways that together influence regenerative growth and result in more or less successful functional recovery. However, these responses can be offset by pathological consequences including neuropathic pain. Calcium signalling plays a major role in the different steps occurring after nerve damage. As part of our studies to unravel the roles of injury-induced molecular changes in dorsal root ganglia (DRG) neurons during their regeneration, we show that the calcium calmodulin kinase CaMK1a is markedly induced in mouse DRG neurons in several models of mechanical peripheral nerve injury, but not by inflammation. Intrathecal injection of NRTN or GDNF significantly prevents the post-traumatic induction of CaMK1a suggesting that interruption of target derived factors might be a starter signal in this de novo induction. Inhibition of CaMK signalling in injured DRG neurons by pharmacological means or treatment with CaMK1a siRNA resulted in decreased velocity of neurite growth in vitro. Altogether, the results suggest that CaMK1a induction is part of the intrinsic regenerative response of DRG neurons to peripheral nerve injury, and is thus a potential target for therapeutic intervention to improve peripheral nerve regeneration.     

Neutralization of Schwann Cell-Secreted VEGF Is Protective to In Vitro and In Vivo Experimental Diabetic Neuropathy

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

激活素促进鸡胚神经节神经突起生长作用

为了探讨激活素(activin)促进鸡胚背根神经节(dorsal root ganglia,DRG)突起生长、维持神经节细胞生存作用及其与一氧化氮(NO)释放的关系,实验采用8 d的鸡胚分离背根神经节,原代培养法,观察鸡胚背根神经节的体外生长情况。研究结果表明,添加激活素A培养的背根神经节有明显的神经突起生长,形成密集的网络,背根神经节可存活8~10 d;而阴性对照组几乎无神经突起生长,背根神经节可存活3~4 d。添加激活素A的背根神经节单层培养神经节细胞也可长期存活;而阴性对照组在培养第5 d几乎无神经节细胞生存。NO检测结果显示,添加激活素A培养的背根神经节上清NO分泌水平明显降低,与阴性对照组比较差异显著(P<0.05);激活素A与神经生长因子(nerve growth factor,NGF)具有协同抑制背根神经节NO分泌作用。激活素结合蛋白(follistatin)明显抑制激活素A诱导的背根神经节神经突起生长。研究结果提示,激活素可维持鸡胚神经节细胞存活并刺激神经突起生长,其作用与抑制神经损伤因子NO的释放有关。     

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.     

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth

Treatment for peripheral nerve injuries includes the use of autografts and nerve guide conduits (NGCs). However, outcomes are limited, and full recovery is rarely achieved. The use of nerve scaffolds as a platform to surface immobilize neurotrophic factors and deliver locally is a promising approach to support neurite and nerve outgrowth after injury. We report on a bioactive surface using functional amine groups, to which heparin binds electrostatically. X-ray photoelectron spectroscopy analysis was used to characterize the presence of nitrogen and sulfur. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were bound by electrostatic interaction to heparin, and the release profile evaluated by enzyme-linked immunosorbent assay, which showed that ca. 1% of NGF was released from each of the bioactive surface within 7 days. Furthermore, each surface showed a maximum release of 97% of BDNF. Neurotrophin release on neurite outgrowth was evaluated by primary dorsal root ganglion with a maximum neurite growth response in vitro of 1,075 µm detected for surfaces immobilized with NGF at 1 ng/ml. In summary, the study reports on the design and construction of a biomimetic platform to deliver NGF and BDNF using physiologically low concentrations of neurotrophin. The platform is directly applicable and scalable for improving the regenerative ability of existing NGCs and scaffolds.     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Directed and enhanced neurite growth with pulsed magnetic field stimulation

Pulsed magnetic field (PMF) stimulation was applied to mammalian neurons in vitro to influence axonal growth and to determine whether induced current would direct and enhance neurite growth in the direction of the current. Two coils were constructed from individual sheets of copper folded into a square coil. Each coil was placed in a separate water-jacketed incubator. One was energized by a waveform generator driving a power amplifier, the other was not energized. Whole dorsal root ganglia (DRG) explant cultures from 15-day Sprague-Dawley rat embryos were established in supplemented media plus nerve growth factor (NGF) at concentrations of 0-100 ng/mL on a collagen-laminin substrate. Dishes were placed at the center of the top and bottom of both coils, so that the DRG were adjacent to the current flowing in the coil. After an initial 12 h allowing DRG attachment to the substrate floor, one coil was energized for 18 h, followed by a postexposure period of 18 h. Total incubation time was 48 h for all DRG cultures. At termination, DRG were histochemically stained for visualization and quantitative analysis of neurite outgrowth. Direction and length of neurite outgrowth were recorded with respect to direction of the current. PMF exposed DRG exhibited asymmetrical growth parallel to the current direction with concomitant enhancement of neurite length. DRG cultures not PMF exposed had a characteristic radial pattern of neurite outgrowth. These results suggest that PMF may offer a noninvasive mechanism to direct and promote nerve regeneration.     

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.

Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.     

The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3-kinase signaling pathway

Nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) play an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Adult DRG neurons exhibit neurotrophin-independent survival, providing an excellent system with which to study trophic factor effects on neurite growth in the absence of significant survival effects. Using young adult rat DRG neurons we have demonstrated a synergistic effect of NGF plus IGF (N + I), compared with either factor alone, in promoting neurite growth. Not only does the presence of NGF and IGF-1 enhance neurite initiation, it also significantly augments the extent of neurite branching and elongation. We have also examined potential mechanism(s) underlying this synergistic effect. Immunoblotting experiments of classical growth factor intermediary signalling pathways (PI 3-K-Akt-GSK-3 and Ras-Raf-MAPK) were performed using phospho-specific antibodies to assess activation state. We found that activation of Akt and MAPK correlated with neurite elongation and branching. However, using pharmacological inhibitors, we observed that a PI 3-K pathway involving both Akt and GSK-3 appeared to be more important for neurite extension and branching than MAPK-dependent signalling. In fact, inhibition of activation of MAPK with U0126 resulted in increased neuritic branching, possibly as a result of the concomitant increase observed in phospho-Akt. Furthermore, inhibition of GSK3 (which is negatively regulated by phosphorylation on S9/S21) also resulted in increased growth. Our data point to signalling convergence upon the PI 3-K-Akt-GSK-3 pathway that underlies the NGF plus IGF synergism. In addition, to our knowledge, this is the first report in primary neurons that inhibition of GSK3 results in an enhanced neurite growth.     

Sildenafil Ameliorates Long Term Peripheral Neuropathy in Type II Diabetic Mice

Diabetic peripheral neuropathy is a common complication of long-standing diabetes mellitus. To mimic clinical trials in which patients with diabetes enrolled have advanced peripheral neuropathy, we investigated the effect of sildenafil, a specific inhibitor of phosphodiesterase type 5 enzyme, on long term peripheral neuropathy in middle aged male mice with type II diabetes. Treatment of diabetic mice (BKS.Cg-m+/+Lepr^db/J, db/db) at age 36 weeks with sildenafil significantly increased functional blood vessels and regional blood flow in the sciatic nerve, concurrently with augmentation of intra-epidermal nerve fiber density in the skin and myelinated axons in the sciatic nerve. Functional analysis showed that the sildenafil treatment considerably improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal stimulus sensitivity compared with the saline treatment. In vitro studies showed that mouse dermal endothelial cells (MDE) cultured under high glucose levels exhibited significant down regulation of angiopoietin 1 (Ang1) expression and reduction of capillary-like tube formation, which were completely reversed by sildenafil. In addition, incubation of dorsal root ganglia (DRG) neurons with conditioned medium harvested from MDE under high glucose levels suppressed neurite outgrowth, where as conditional medium harvested from MDE treated with sildenafil under high glucose levels did not inhibit neurite outgrowth of DRG neurons. Moreover, blockage of the Ang1 receptor, Tie2, with a neutralized antibody against Tie2 abolished the beneficial effect of sildenafil on tube formation and neurite outgrowth. Collectively, our data indicate that sildenafil has a therapeutic effect on long term peripheral neuropathy of middle aged diabetic mice and that improvement of neurovascular dysfunction by sildenafil likely contributes to the amelioration of nerve function. The Ang1/Tie2 signaling pathway may play an important role in these restorative processes.     

microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection

Dorsal root ganglia (DRG) neurons spontaneously undergo neurite growth after nerve injury. MicroRNAs (miRNAs), as small, non-coding RNAs, negatively regulate gene expression in a variety of biological processes. The roles of miRNAs in the regulation of responses of DRG neurons to injury stimuli, however, are not fully understood. Here, microarray analysis was performed to profile the miRNAs in L4-L6 DRGs following rat sciatic nerve transection. The 26 known miRNAs were differentially expressed at 0, 1, 4, 7, 14 d post injury, and the potential targets of the miRNAs were involved in nerve regeneration, as analyzed by bioinformatics. Among the 26 miRNAs, microRNA-222 (miR-222) was our research focus because its increased expression promoted neurite outgrowth while it silencing by miR-222 inhibitor reduced neurite outgrowth. Knockdown experiments confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major inhibitor of nerve regeneration, was a direct target of miR-222 in DRG neurons. In addition, we found that miR-222 might regulate the phosphorylation of cAMP response element binding protein (CREB) through PTEN, and c-Jun activation might enhance the miR-222 expression. Collectively, our data suggest that miR-222 could regulate neurite outgrowth from DRG neurons by targeting PTEN.